Sucrose Feedstock Utilization for Oil-Based Fuel Manufacturing

• US 20090035842A1
• Filed: 06/02/2008
• Published: 02/05/2009
• 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 8,476,059 B2
• Time in Patent Office: Days
• US Class Current: 435/254.220
• 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 manufacturing oils and oil-based products such as transportation fuels, industrial chemicals, edible oils, lubricants and plastics using sugar cane, sugar beets, and cane/beet agricultural processing byproducts as a feedstock for bioproduction processes. The disclosed processes utilize oil-bearing microbes as a conversion technology to convert chemical energy produced by sugar cane and sugar beets into energy-containing oils and oil derivatives. Also provided herein are oil-bearing microbes containing one or more exogenous sucrose utilization genes.

Claims

1. A microorganism containing an exogenous sucrose utilization gene.

2. The microorganism of claim 1, wherein the gene encodes a sucrose transporter.

3. The microorganism of claim 1, wherein the gene encodes a sucrose invertase.

4. The microorganism of claim 1, wherein the gene encodes a fructokinase.

5. The microorganism of claim 1, wherein the microorganism is a microalgae.

6. The microorganism of claim 5, wherein the microorganism is selected from the group consisting of the microalgae listed in Table 1.

7. The microorganism of claim 6, wherein the microorganism is a species of the genus Chlorella.

8. The microorganism of claim 7, wherein the microorganism is a species selected from the group consisting of Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides.

9. A cell of the species Chlorella protothecoides, Chlorella emersonii, or Chlorella minutissima wherein the cell contains an exogenous gene.

10. The cell of claim 9, wherein the exogenous gene encodes a protein selected from the group consisting of a sucrose transporter, a sucrose invertase, a lipid modification enzyme, a hydrocarbon modification enzyme and a fructokinase.

11. The cell of claim 10, wherein the protein is a sucrose invertase secreted into the extracellular space.

12. The cell of claim 10, wherein the protein is a sucrose invertase targeted to the cytoplasm.

13. A microbial culture containing:

(a) a population of microorganisms; and

(b) a culture medium comprising (i) sucrose, and (ii) a sucrose invertase enzyme.

14. The microbial culture of claim 13, wherein the microorganisms are microalgae.

15. The microbial culture of claim 14, wherein the microorganisms are selected from the group consisting of the microalgae listed in Table 1.

16. The microbial culture of claim 15, wherein the microorganisms are a species of the genus Chlorella.

17. The microbial culture of claim 16, wherein the microorganisms are selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, ChIorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, and Chlorella zofingiensis.

18. The microbial culture of claim 13, wherein the microorganisms are oleaginous yeast.

19. The microbial culture of claim 18, wherein the oleaginous yeast are selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica.

20. The microbial culture of claim 13, wherein the microorganisms are fungi.

21. The microbial culture of claim 20, wherein the fungi are 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 iulacinum, 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.

22. The microbial culture of claim 13, wherein the microorganisms contain at least one exogenous sucrose utilization gene.

23. The microbial culture of claim 22, wherein the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.

24. The microbial culture of claim 23, wherein the sucrose utilization gene encodes a sucrose transporter.

25. The microbial culture of claim 13, wherein the sucrose invertase enzyme is a secretable sucrose invertase enzyme encoded by an exogenous sucrose invertase gene expressed by the population of microorganisms.

26. The microbial culture of claim 13, wherein the microorganisms contain at least one exogenous gene encoding a lipid pathway enzyme.

27. The microbial culture of claim 26, wherein the lipid pathway enzyme is selected from the group consisting of a fatty acyl-ACP thioesterases, a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase.

28. A microbial culture containing:

(a) a population of microorganisms; and

(b) a culture medium comprising (i) molasses, and (ii) a sucrose invertase enzyme.

29. The microbial culture of claim 28, wherein the microorganisms are microalgae.

30. The microbial culture of claim 29, wherein the microorganisms are selected from the group consisting of the microalgae listed in Table 1.

31. The microbial culture of claim 30, wherein the microorganisms are a species of the genus Chlorella.

32. The microbial culture of claim 31, wherein the microorganisms are selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vannielli, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, and Chlorella zofingiensis.

33. The microbial culture of claim 28, wherein the microorganisms are oleaginous yeast.

34. The microbial culture of claim 33, wherein the oleaginous yeast are selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica.

35. The microbial culture of claim 28, wherein the microorganisms are fungi.

36. The microbial culture of claim 35, wherein the fungi are 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.

37. The microbial culture of claim 28, wherein the microorganisms contain at least one exogenous sucrose utilization gene.

38. The microbial culture of claim 37, wherein the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.

39. The microbial culture of claim 38, wherein the sucrose utilization gene encodes a sucrose transporter.

40. The microbial culture of claim 28, wherein the sucrose invertase enzyme is a secretable sucrose invertase enzyme encoded by an exogenous sucrose invertase gene expressed by the population of microorganisms.

41. The microbial culture of claim 28, wherein the microorganisms contain at least one exogenous gene encoding a lipid pathway enzyme.

42. The microbial culture of claim 41, wherein the lipid pathway enzyme is selected from the group consisting of a fatty acyl-ACP thioesterases, a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase.

43. A microbial culture containing:

(a) a population of microorganisms; and

(b) a culture medium comprising (i) sucrose;

(ii) lignin; and

(iii) a sucrose invertase enzyme.

44. The microbial culture of claim 43, wherein the microorganisms are microalgae.

45. The microbial culture of claim 44, wherein the microorganisms are selected from the group consisting of the microalgae listed in Table 1.

46. The microbial culture of claim 45, wherein the microorganisms are a species of the genus Chlorella.

47. The microbial culture of claim 46, wherein the microorganisms are selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorellafusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, and Chlorella zofingiensis.

48. The microbial culture of claim 43, wherein the microorganisms are oleaginous yeast.

49. The microbial culture of claim 48, wherein the oleaginous yeast are selected from the group consisting of Cryptococcus curvatus, Cryptococcus terricolus, Candida sp., Lipomyces starkeyi, Lipomyces lipofer, Endomycopsis vernalis, Rhodotorula glutinis, Rhodotorula gracilis, and Yarrowia lipolytica.

50. The microbial culture of claim 43, wherein the microorganisms are fungi.

51. The microbial culture of claim 50, wherein the fungi are 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.

52. The microbial culture of claim 43, wherein the microorganisms contain at least one exogenous sucrose utilization gene.

53. The microbial culture of claim 52, wherein the sucrose utilization gene encodes a sucrose transporter, a sucrose invertase, a hexokinase, a glucokinase, or a fructokinase.

54. The microbial culture of claim 53, wherein the sucrose utilization gene encodes a sucrose transporter.

55. The microbial culture of claim 43, wherein the sucrose invertase enzyme is a secretable sucrose invertase enzyme encoded by an exogenous sucrose invertase gene expressed by the population of microorganisms.

56. The microbial culture of claim 43, wherein the microorganisms contain at least one exogenous gene encoding a lipid pathway enzyme.

57. The microbial culture of claim 56, wherein the lipid pathway enzyme is selected from the group consisting of a fatty acyl-ACP thioesterase, a stearoyl-ACP desaturase, a glycerolipid desaturase, a pyruvate dehydrogenase, an acetyl-CoA carboxylase, an acyl carrier protein, and a glycerol-3 phosphate acyltransferase.

58. (canceled)

59. A method of cultivating microalgae, comprising:

(a) providing a microalgae cell capable of heterotrophic growth; and

(b) placing the microalgae cell in culture medium comprising sucrose,

wherein the microalgae cell accumulates at least 10% of its dry cell weight as lipid.

60. The method of claim 59, wherein the microalgae cell contains an exogenous sucrose utilization gene.

61. The method of claim 60, wherein the sucrose utilization gene encodes a sucrose invertase.

62. The method of claim 59, wherein a sucrose invertase that is not produced by the microalgae cell is added to the culture medium.

63. The method of claim 59, wherein the microalgae cell is genetically engineered to express a secretable sucrose invertase enzyme.

64. The method of claim 59, wherein the microalgae cell is a species of the genus Chlorella.

65. The method of claim 64, wherein the microalgae cell is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorella fusca, Chlorella fusca var. vacuolata, Chlorella glucotropha, Chlorella infusionum, Chlorella infusionum var. Actophila, Chlorella infusionum var. Auxenophila, Chlorella kessleri, Chlorella luteoviridis, Chlorella luteoviridis var. aureoviridis, Chlorella luteoviridis var. Lutescens, Chlorella miniata, Chlorella minutissima, Chlorella mutabilis, Chlorella nocturna, Chlorella parva, Chlorella photophila, Chlorella pringsheimii, Chlorella protothecoides, Chlorella regularis, Chlorella regularis var. minima, Chlorella regularis var. umbricata, Chlorella reisiglii, Chlorella saccharophila, Chlorella saccharophila var. ellipsoidea, Chlorella salina, Chlorella simplex, Chlorella sorokiniana, Chlorella sp., Chlorella sphaerica, Chlorella stigmatophora, Chlorella vanniellii, Chlorella vulgaris, Chlorella vulgaris, Chlorella vulgaris f. tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, and Chlorella zofingiensis.

66. The method of claim 59, wherein the culture medium further comprises at least one additional carbon source selected from the group consisting of 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 and fiber sorghum.

67. The method of claim 59, wherein the lipid comprises at least 50% C18:

1.