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1. (WO2013158938) TAILORED OILS
ملاحظة: نص مبني على عمليات التَعرف الضوئي على الحروف. الرجاء إستخدام صيغ PDF لقيمتها القانونية

WHAT IS CLAIMED IS:

1. An oleaginous microalgal cell, optionally comprising 23 S rRNA having at least 65% nucleotide sequence identity to SEQ ID NO: 76, optionally obligately

heterotrophic, and optionally comprising an exogenous sucrose invertase gene so that the cell can grow on sucrose as a sole carbon source, the cell comprising an exogenous gene encoding an active LPAAT enzyme, the cell producing an oil comprising triglycerides, wherein the oil is, by virtue of the LPAAT activity:

(a) enriched in triglycerides with midchain fatty acids; or

(b) enriched in triglycerides of the saturated-unsaturated-saturated type.

2. The cell of claim 1, wherein the triglycerides of the oil comprise 40, 50, 60, 70, or 80% or more of C8:0, C10:0, C12:0, C14:0, or C16:0 fatty acids.

3. The cell of claim 1 or 2, wherein the cell further comprises an exogenous gene encoding an active FATB acyl-ACP thioesterase.

4. The cell of claim 3, wherein the triglycerides of the oil are enriched in midchain fatty acids by greater than 70% as a result of the expression of the exogenous LPAAT and acyl-ACP thioesterase.

5. The cell of any one of claims 1 to 4, wherein the cell further comprises recombinant nucleic acids operable to encode an exogenous KAS I or KAS IV enzyme or to reduce the activity of an endogenous KAS I enzyme.

6. The cell of any one of claims 1 to 5, wherein the cell further comprises nucleic acids operable to reduce the expression of a delta 12 fatty acid desaturase, optionally via a regulatable promoter, so as to produce an oil with linoleic and linolenic acids totaling 5 area percent or less by FAME GC/FID.

7. The cell of claim 1, wherein the oil is enriched in triglycerides of the saturated-unsaturated-saturated type.

8. The cell of claim 7, wherein the oil is enriched in SOS, POS, and/or POP.

9. The cell of claim 8, wherein the oil comprises triglycerides comprising at least 50% SOS, and optionally less than 10% SSS.

10. The cell of claim 8 or 9, further comprising a knockout or knockdown of a stearoyl-ACP desaturase gene, FatA gene, or both.

11. The cell of any of claims 7 to 9, further comprising recombinant nucleic acids operable to increase beta-ketoacyl CoA synthase activity.

12. The cell of claim 11, wherein the nucleic acids operable to increase beta-ketocyl synthase activity comprise an exogenous gene encoding a beta-ketoacyl CoA synthase.

13. The cell of any one of claims 7 to 12, wherein the ratio of stearate to oleate in oil is 3:1 + 20%.

14. The cell of any one of claims 7 to 13, wherein POP, SOS, and POS in the oil comprise at least 30% in sum.

15. The cell of any one of claims 7 to 14, wherein the oil comprises at least

30% POS.

16. The cell of any one of claims 7 to 15, wherein the oil comprises POP at 16% ±20%, POS at 38%+20%, and SOS at 23% + 20%.

17. The cell of any one of claims 7 to 16, wherein the fatty acid profile of the oil comprises 1 to 4% arachidic acid.

18. The cell of any one of claims 7 to 17, wherein the cell further comprises nucleic acids operable to reduce the expression of a delta 12 fatty acid desaturase, optionally via a regulatable promoter, so as to produce an oil with linoleic and linolenic acids totaling 5 area percent or less.

19. The cell of any one of claims 7 to 17, wherein the oil has greater than 65% SOS, less than 45% unsaturated fatty acid, less than 5% polyunsaturated fatty acids, less than 1% lauric acid, and less than 2% trans fatty acids.

20. The cell of any one of claims 1 to 19, wherein the LP A AT has the amino acid sequence of SEQ ID NO: 78 or SEQ ID NO: 79 or a sequence having at least 95% identity to SEQ ID NO:78 or SEQ ID NO: 79.

21. A method for producing an oil comprising providing or cultivating a cell according to any one of claims 1 to 20, and extracting the oil, the cell optionally being cultivated heterotrophically.

22. An oil comprising triglycerides produced according to claim 21.

23. The oil of claim 22, comprising one or more of:

at least 10% ergosterol;

ergosterol and β-sitosterol, wherein the ratio of ergosterol to β-sitosterol is greater than 25:1;

ergosterol and brassicasterol;

ergosterol, brassicasterol, and poriferasterol;

and wherein the oil is optionally free from one or more of β-sitosterol, campesterol, and stigmasterol.

24. The oil of claim 22 wherein the oil forms β polymorph crystals.

25. The oil of claim 24, wherein the crystals have a 2L lamellae structure.

26. The oil of claim 24, wherein the crystals have a 3L lamellae structure.

27. The oil of claim 22, wherein the oil forms β' polymorph crystals.

28. The oil of claim 24, wherein the crystals have a 2L lamellae structure.

29. The oil of claim 24, wherein the crystals have a 3L lamellae structure.

30. The oil of any one of claims 22 to 29, wherein the triglycerides have a fatty acid profile characterized in that the sum of the percentage of stearate and palmitate is equal to the percentage of oleate multiplied by 2.0 +/- 40%.

31. The oil of any one of claims 22 to 30, wherein the oil has greater than 65% SOS triglycerides, less than 45% unsaturated fatty acid, less than 5% unsaturated fatty acids, less than 1% lauric acid, and less than 2% trans fatty acid.

32. The oil of any one of claims 22 to 31, wherein the sum of the percent stearate and palmitate in the fatty acid profile is twice the percentage of oleate, + 20% .

33. The oil of claim 32, wherein the sn-2 profile has at least 40% oleate.

34. The oil of any one of claims 22 to 33, wherein the oil is at least 40, 50, 60, 70, 80, or 90% SOS.

35. The oil of any one of claims 22 to 33, wherein the oil is a roll-in shortening having a melting temperature of between 30°C and 40°C.

36. The oil of claim 35, comprising β' polymorphic crystals.

37. The oil of claim 35 or 36, having a solid fat content of less than 15% at

35°C.

38. The oil of any one of claims 35 to 37, comprising 15 to 20% C8 to C14 fatty acids, 45-50% fatty acids of C16 and higher, and/or 30-25% unsaturated fatty acids.

39. A food, fuel or chemical product produced using the oil of any one of claims 22 to 38.

40. A natural oil or RBD oil produced from a natural oil, the oil comprising 3.5% or less saturated fatty acids, and optionally comprising greater than 50% oleic acid and/or greater than 1 % palmitoleic acid.

41. The oil of claim 40, having between 0.1 and 3.5% saturated fatty acids.

42. The oil of claim 40 or 41, comprising at least 90% oleic acid.

43. The oil of claim 42, comprising at least 3% polyunsaturated fatty acids.

44. An oleaginous cell, optionally comprising 23 S rRNA having at least 65% nucleotide sequence identity to SEQ ID NO: 76 and optionally obligately heterotrophic, that produces an oil comprising 3.5% or less saturated fatty acids.

45. The cell of claim 44, wherein the cell is of the genus Prototheca.

46. The cell of claim 44, further comprising a FATA knockout or knockdown.

47. The cell of any one of claims 44 to 46, comprising an exogenous gene encoding an enzyme active to desaturate palmitoyl-CoA to plamitoyl-CoA.

48. The cell of claim 47, wherein the exogenous gene is a PAD gene.

49. The cell of claim 47, wherein the exogenous gene is a SAD gene having desaturase activity toward palmitoyl-ACP.

50. The cell of any one of claims 44 to 49, further comprising an

overexpressed KASII enzyme.

51. The cell of any one of claims 44 to 50, further comprising nucleic acids operable to reduce the expression of a delta 12 fatty acid desaturase, optionally via a regulatable promoter, so as to produce an oil with linoleic and linolenic acids totaling 5 area percent or less.

52. An oil produced by the cell of any one of claims 44 to 51, optionally refined, bleached and deodorized, the oil comrpising one or more of:

at least 10% ergosterol;

ergosterol and b-sitosterol, wherein the ratio of ergosterol to b-sitosterol is greater than 25:1;

ergosterol and brassicasterol; and

ergosterol, brassicasterol, and poriferasterol;

wherein the oil is optionally free from one or more of -sitosterol, campesterol, and stigmasterol.

53. A method for producing an oil having 3.5% or less saturated fatty acids, the method comprising providing or cultivating a cell of any one of claims 5 to 12, and extracting the oil from the cell.

54. A method for producing a food comprising incorporating an oil produced by the method of claim 53 into the food, wherein the finished food product has 3.5% or less saturated fat.

55. A recombinant oleaginous cell, optionally comprising 23S rRNA having at least 65% nucleotide sequence identity to SEQ ID NO: 76 and optionally obligately heterotrophic, the cell comprising an exogenous gene encoding an active ketoacyl-CoA synthase enzyme.

56. The cell of claim 55, wherein the cell produces an oil comprising greater than 20% erucic acid.

57. A cell according to claim 56, wherein the cell produces an oil comprising greater than 60% erucic acid.

58. The cell of any one of claims 55 to 57, wherein the cell comprises at least

40% oil.

59. The cell of any one of claims 55 to 58, wherein the cell is of the genus Prototheca, and optionally of the species Prototheca moriformis.

60. An oil produced by the cell of any one of claims 55 to 59, comprising one or more of:

at least 10% ergosterol;

ergosterol and β-sitosterol, wherein the ratio of ergosterol to β-sitosterol is greater than 25: 1 ;

ergosterol and brassicasterol; and

ergosterol, brassicasterol, and poriferasterol;

wherein the oil is optionally free from one or more of β-sitosterol, campesterol, and stigmasterol.

61. A chemical produced from the oil of claim 60.

62. A method for producing an oil comprising providing or cultivating a cell according to any one of claims 55 to 59 and extracting an oil from the cell.

63. A recombinant oleaginous cell comprising recombinant nucleic acids operable to suppress the activity of a delta 12 fatty acid desaturase gene product so that the cell produces an oil with a triacylglycerol profile having less than 5% linoleic acid.

64. The cell of claim 63, wherein the cell produces an oil with a triacylglycerol profile having less than 3% linoleic acid.

65. The cell of claim 64, wherein the cell produces an oil with a triacylglycerol profile having less than 2% linoleic acid.

66. The cell of claim 63, wherein the cell is a linoleic acid auxotroph or activity of the delta 12 fatty acid desaturase can be suppressed via environmental conditions so as to produce the oil.

67. The cell of claim 66, wherein the delta 12 fatty acid desaturase is regulatable via environmental conditions due to a regulatable promoter in operable linkage to the delta 12 fatty acid desaturase gene.

68. The cell of claim 67, wherein the regulatable promoter is regulatable by change in media pH or nitrogen levels.

69. The cell of any one of claims 64 to 68, further comprising recombinant nucleic acids operable to express an exogenous KASII, LPAAT, or FATB enzyme.

70. The cell of any one of claims 64 to 69, further comprising recombinant nucleic acids operable to knockout or knockdown the expression of a stearoyl ACP desaturase enzyme.

71. The cell of any one of claims 64 to 70, further comprising recombinant nucleic acids operable to knockout or knockdown the expression of an endogenous FatA-encoded acyl-ACP thioesterase.

72. The cell of any one of claims 64 to 71, wherein the oil is stable at 110°C so that the inflection point in conductance is not yet reached by 20 hours under conditions of the AOCS Cd 12b-92 Rancimat test.

73. The cell of any one of claims 64 to 71, wherein the oil is stable at 110°C so that the inflection point in conductance is not yet reached by 5 days under conditions of the AOCS Cd 12b-92 Rancimat test, when 1050ppm of tocopherol and 500 pm of ascorbyl palmitate are added to the oil.

74. A method comprising:

(a) providing a recombinant oleaginous cell, optionally comprising 23 S rRNA having at least 65% nucleotide sequence identity to SEQ ID NO:76, optionally obligately heterotrophic, the cell comprising recombinant nucleic acids operable to modify the amount of a fatty acid made by the cell in response to a change in an environmental condition;

(b) cultivating the cell under a first environmental condition that is permissive to synthesis of the fatty acid so as to allow for cell division and increase the number of cells; (c) cultivating the cell under a second environmental condition that, due to the recombinant nucleic acids, reduces the synthesis of the fatty acid and thus the amount of that fatty acid in an oil produced by the cell; and

(d) extracting the oil from the cell.

75. The method of claim 74, wherein the cell comprises exogenous nucleic acids operable to reduce the activity of a delta 12 fatty acid desaturase so as to reduce the amount of linoleic acid in the oil.

76. The method of claim 74 or 75, wherein the linoleic acid is depleted in the oil by at least than 50, 60, 70, 80, or 90%.

77. The method of any one of claims 74 to 76 wherein the cell is cultivated heterotrophically.

78. The method of any one of claims 74 to 77, wherein the cell is a microalgal cell.

79. The method of any one of claims 74 to 78, wherein the cell produces at least 40, 50, 60, 70, 80, or 90% oil by dry cell weight.

80. The method of any one of claims 74 to 79, wherein the first environmental condition is a first pH and the second environmental condition is a second pH of cultivation media.

81. The method of any one of claims 74 to 80, wherein, when extracted from the cell, the oil is stable at 110°C so that the inflection point in conductance is not yet reached by 20 hours under conditions of the AOCS Cd 12b-92 Rancimat test.

82. The method of any one of claims 74 to 81, wherein when extracted from the cell, the oil is stable at 110°C so that the inflection point in conductance is not yet reached by 5 days under conditions of the AOCS Cd 12b-92 Rancimat test, when 1050ppm of tocopherol and 500 pm of ascorbyl palmitate are added to the oil.

83. The method of any one of claims 74 to 82, wherein the cell comprises an exogenous gene encoding a KAS II enzyme and optionally a knockout or knockdown of a FatA gene.

84. The method of any one of claims 74 to 83, wherein the recombinant nucleic acids operable to modify the amount of a fatty acid made by the cell comprise an inhibitory RNA targeting a FAD gene, the production of the inhibitory RNA being under control of a regulatable promoter.

85. The method of claim 83 or 84, wherein the oil is characterized by a fatty acid profile with greater than 60% oleic acid and less than 3% polyunsaturates.

86. The method of claim 85, wherein the oil is characterized by a fatty acid profile with greater than 70% oleic acid and less than 2% polyunsaturates.

87. The method of claim 86, wherein the oil is characterized by a fatty acid profile with greater than 80% oleic acid and less than 1% polyunsaturates.

88. An oil produced by the method of any one of claims 74 to 87.

89. The oil of claim 88 comprising 0.01 to 2% linoleic acid and (i) 80 to 95% oleic acid or (ii) more than 40% of C8, CIO, C12, C14 or C16 fatty acids.

90. The oil of claim 88 or 89, further comprising one or more of: at least 10% ergosterol;

ergosterol and b-sitosterol, wherein the ratio of ergosterol to b-sitosterol is greater than 25:1;

ergosterol and brassicasterol; and

ergosterol, brassicasterol, and poriferasterol.

91. A product produced from the oil of any one of claims 88 to 90.

92. The product of claim 91, wherein the product is a food, fuel or chemical.

93. The product of claim 91, wherein the product is a frying oil, lubricating oil, cleaning solvent, surfactant, foam or lubricant.

94. The product of claim 91, wherein the product is an oleic acid dimer.

95. A construct, vector, chromosome or host cell comprising nucleic acids encoding a protein having at least 90% identity to SEQ ID NOs: 77 to 79.

96. The construct, vector, chromosome or host cell of claim 95, encoding a protein having at least 95% identity to SEQ ID NOs: 77 to 79.

97. The construct, vector, chromosome or host cell of claim 95, encoding a protein having at least 98% identity to SEQ ID NOs: 77 to 79.

98. The construct, vector, chromosome, or host cell of any of claims 95 to 97, comprising nucleic acids having at least 80, 90, 95, 96, 97, 98, or 99% sequence identity to SEQ ID NOs:80-85 or equivalent sequences by virtue of the degeneracy of the genetic code.