Lipid Pathway Modification in Oil-Bearing Microorganisms

• US 20090061493A1
• Filed: 06/02/2008
• Published: 03/05/2009
• 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:
  US12/131,804
• Publication Number:
  US 20090061493A1
• Time in Patent Office: Days
• US Class Current: 435/157
• 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 modifying the lipids produced by microbial organisms through genetic engineering. The invention also provides genetically engineered microbes and methods of fermenting microbes for oil production. Also provided are oils, fuels, oleochemicals, chemical precursors, and other compounds manufactured by such modified microorganisms. Exemplary oil-bearing organisms include organisms containing one or more exogenous genes encoding a fatty acyl-ACP thioesterase, fatty acyl-CoA/aldehyde reductase, fatty acyl-CoA reductase, fatty aldehyde reductase, fatty aldehyde decarbonylase, and/or an acyl carrier protein.

Claims

1. A microalgae or yeast cell that has been genetically engineered and/or selected to express a lipid pathway enzyme at an altered level compared to a wild-type cell of the same species.

2. The cell of claim 1, wherein the cell produces more lipid compared to the wild-type cell when both cells are grown under the same conditions.

3. The cell of claim 1, wherein the cell has been genetically engineered and/or selected to express a lipid pathway enzyme at a higher level than the wild-type cell.

4. The cell of claim 3, wherein the lipid pathway enzyme is selected from the group consisting of pyruvate dehydrogenase, acetyl-CoA carboxylase, acyl carrier protein, and glycerol-3 phosphate acyltransferase.

5. The cell of claim 1, wherein the cell has been genetically engineered and/or selected to express a lipid pathway enzyme at a lower level than the wild-type cell.

6. The cell of claim 5, wherein the lipid pathway enzyme comprises citrate synthase.

7. The cell of claim 1, wherein the cell has been genetically engineered and/or selected to express a global regulator of fatty acid synthesis at an altered level compared to the wild-type cell, whereby the expression levels of a plurality of fatty acid synthetic genes are altered compared to the wild-type cell.

8. The cell of claim 1, wherein the lipid pathway enzyme comprises an enzyme that modifies a fatty acid.

9. The cell of claim 8, wherein the lipid pathway enzyme is selected from a stearoyl-ACP desaturase and a glycerolipid desaturase.

10. The cell of claim 1, wherein the cell is a microalgae species selected from Table 1.

11. The microalgae cell of claim 10, wherein the microalgae is of the genus Chlorella.

12. The microalgae cell of claim 11, wherein the microalgae is of the species 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 pyrenoidosa, 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. vulgarisf tertia, Chlorella vulgaris var. vulgaris f. viridis, Chlorella xanthella, and Chlorella zofingiensis.

13. An oil-producing microbe containing one or more exogenous genes, wherein the exogenous genes encode protein(s) selected from the group consisting of a fatty acyl-ACP thioesterase, a fatty acyl-CoA reductase, a fatty aldehyde reductase, a fatty acyl-CoA/aldehyde reductase, a fatty aldehyde decarbonylase, and an acyl carrier protein.

14. The microbe of claim 13, wherein the microbe is a microalgae.

15. The microbe of claim 13, wherein the microbe is an oleaginous yeast.

16. The microbe of claim 15, 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, and Yarrowia lipolytica.

17. The microbe of claim 13, wherein the microbe is a fungus.

18. The microbe of claim 17, wherein the 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.

19. The microbe of claim 13, wherein the microbe is selected from the group consisting of the microorganisms listed in Table 1.

20. The microbe of claim 19, wherein the microbe is a species of the genus Chlorella.

21. The microbe of claim 20, wherein the species 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 pyrenoidosa, 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 vulgarisf 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.

22. The microbe of claim 21, wherein the species is Chlorella protothecoides, Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, or Chlorella sp.

23. The microbe of claim 13, wherein the exogenous gene is in operable linkage with a promoter, which is inducible or repressible in response to a stimulus.

24. The microbe of claim 23, wherein the stimulus is selected from the group consisting of an exogenously provided small molecule, heat, cold, and light.

25. The microbe of claim 13, wherein the exogenous gene is expressed in a cellular compartment.

26. The microbe of claim 25, wherein the cellular compartment is selected from the group consisting of a chloroplast and a mitochondrion.

27. The microbe of claim 13, wherein the exogenous gene encodes a fatty acid acyl-ACP thioesterase.

28. The microbe of claim 27, wherein the thioesterase encoded by the exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty acid from an acyl carrier protein (ACP).

29. The microbe of claim 28, wherein the thioesterase encoded by the exogenous gene catalyzes the cleavage of a 10 to 14-carbon fatty acid from an ACP.

30. The microbe of claim 29, wherein the thioesterase encoded by the exogenous gene catalyzes the cleavage of a 12-carbon fatty acid from an ACP.

31. The microbe of claim 13, wherein the exogenous gene encodes a fatty acyl-CoA/aldehyde reductase.

32. The microbe of claim 31, wherein the reductase encoded by the exogenous gene catalyzes the reduction of a 20 to 30-carbon fatty acyl-CoA to a corresponding primary alcohol.

33. The microbe of claim 31, wherein the reductase encoded by the exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding primary alcohol.

34. The microbe of claim 33, wherein the reductase encoded by the exogenous gene catalyzes the reduction of a 10 to 14-carbon fatty acyl-CoA to a corresponding primary alcohol.

35. The microbe of claim 34, wherein the reductase encoded by the exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol.

36. The microbe of claim 13, wherein the exogenous gene encodes a fatty acyl-CoA reductase.

37. The microbe of claim 36, wherein the reductase encoded by the exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding aldehyde.

38. The microbe of claim 37, wherein the reductase encoded by the exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanal.

39. The microbe of claim 13, wherein the microbe further contains one or more exogenous sucrose utilization genes.

40-62. -62. (canceled)

63. A method of producing a molecule in a microbe population, the method comprising culturing a population of microbes in a culture medium, wherein the microbes contain:

(i) a first exogenous gene encoding a fatty acyl-ACP thioesterase, and

(ii) a second exogenous gene encoding a fatty acyl-CoA/aldehyde reductase; and

wherein the microbes synthesize a fatty acid linked to an acyl carrier protein (ACP), the fatty acyl-ACP thioesterase catalyzes the cleavage of the fatty acid from the ACP to yield, through further processing, a fatty acyl-CoA, and the fatty acyl-CoA/aldehyde reductase catalyzes the reduction of the acyl-CoA to an alcohol.

64. The method of claim 63, wherein the microbe is a microalgae.

65. The method of claim 63, wherein the microbe is an oleaginous yeast.

66. The method of claim 65, 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, and Yarrowia lipolytica.

67. The method of claim 63, wherein the microbe is a fungus.

68. The method of claim 67, wherein the 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.

69. The method of claim 63, wherein the microbe is selected from the group consisting of the microorganisms listed in Table 1.

70. The method of claim 69, wherein the microbe is a species of the genus Chlorella.

71. The method of claim 70, wherein the species is selected from the group consisting of Chlorella anitrata, Chlorella antarctica, Chlorella aureoviridis, Chlorella candida, Chlorella capsulata, Chlorella desiccata, Chlorella ellipsoidea, Chlorella emersonii, Chlorellafusca, Chlorellafusca 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 pyrenoidosa, 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 vulgarisf tertia, Chlorella vulgaris var. airidis, Chlorella vulgaris var. vulgaris, Chlorella vulgaris var. vulgaris f. tertia, Chlorella vulgaris var. vulgarisf viridis, Chlorella xanthella, and Chlorella zofingiensis.

72. The method of claim 71, wherein the species is Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp. or Chlorella protothecoides.

73. The method of claim 63, wherein the culture medium contains glycerol.

74. The method of claim 73, wherein the glycerol is a byproduct of a transesterification process.

75. The method of claim 73, wherein the culture medium contains glycerol and at least one other fixed carbon source.

76. The method of claim 75, wherein the at least one other fixed carbon source is sucrose.

77. The method of claim 75, wherein all of the glycerol and all of the at least one other fixed carbon source are provided to the microbes at the beginning of fermentation.

78. The method of claim 75, wherein the glycerol and the at least one other fixed carbon source are fed to the microbes at a predetermined rate over the course of fermentation.

79. The method of claim 75, wherein:

(a) glycerol is provided to the microbes in the absence of the at least one other fixed carbon source for a first period of time;

(b) the at least one other fixed carbon source is provided at the end of the first period of time; and

(c) the microbes are cultured for a second period of time in the presence of the at least one other fixed carbon source.

80. The method of claim 63, wherein the exogenous genes are in operable linkage with a promoter that is inducible in response to a first stimulus, and wherein the method further comprises:

providing the first stimulus; and

incubating the population of microbes for a first period of time in the presence of the first stimulus to produce an alcohol.

81. The method of claim 80, further comprising extracting the alcohol from aqueous biomass.

82. The method of claim 63, wherein the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 8 to 18-carbon fatty acid from the ACP, and the reductase encoded by the second exogenous gene catalyzes the reduction of an 8 to 18-carbon fatty acyl-CoA to a corresponding primary alcohol, wherein the thioesterase and the reductase act on the same carbon chain length.

83. The method of claim 82, wherein the thioesterase encoded by the first exogenous gene catalyzes the cleavage of an 10 to 14-carbon fatty acid from the ACP, and the reductase encoded by the second exogenous gene catalyzes the reduction of an 10 to 14-carbon fatty acyl-CoA to a corresponding primary alcohol, wherein the thioesterase and the reductase act on the same carbon chain length.

84. The method of claim 83, wherein the thioesterase encoded by the first exogenous gene catalyzes the cleavage of a 12-carbon fatty acid from the ACP, and the reductase encoded by the second exogenous gene catalyzes the reduction of a 12-carbon fatty acyl-CoA to dodecanol.

85. The method of claim 63, wherein the microbes further contain a third exogenous gene encoding an acyl carrier protein.

86. The method of claim 85, wherein the third exogenous gene encodes an acyl carrier protein that is naturally co-expressed with the fatty acyl-ACP thioesterase.

87-135. -135. (canceled)

136. A microalgae cell containing an exogenous gene, wherein the exogenous gene encodes a protein selected from the group consisting of a lipase, a sucrose transporter, a sucrose invertase, a fructokinase, or a polysaccharide-degrading enzyme.

137. The microalgae cell of claim 136, wherein the cell is selected from the microorganisms in Table 1.

138. The microalgae cell of claim 137, wherein the cell is a species of the genus Chlorella.

139. The microalgae cell of claim 138, wherein the species 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, Chlorellci 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 pyrenoidosa, 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.

140. The microalgae cell of claim 139, wherein the cell is Chlorella minutissima, Chlorella emersonii, Chlorella sorokiniana, Chlorella ellipsoidea, Chlorella sp., or Chlorella protothecoides.

141. The microalgae cell of claim 136, wherein the exogenous gene is in operable linkage with a promoter.

142. The microalgae cell of claim 141, wherein the promoter is inducible or repressible in response to a stimulus.

143. The microalgae cell of claim 142, wherein the stimulus is selected from the group consisting of an exogenously provided small molecule, heat, cold, and light.

144. The microalgae cell of claim 136, wherein the exogenous gene is expressed in a cellular compartment.

145. The microalgae cell of claim 144, wherein the cellular compartment is selected from the group consisting of a chloroplast and a mitochondrion.

148. The microalgae cell of claim 136, wherein the gene encodes a polysaccharide-degrading enzyme.

149. The microalgae cell of claim 148, wherein the polysaccharide-degrading enzyme is endogenous to a Chlorella virus.

146-147. -147. (canceled)

150-162. -162. (canceled)