REDUCTION OF THE TOXIC EFFECT OF IMPURITIES FROM RAW MATERIALS BY EXTRACTIVE FERMENTATION

• US 20090084025A1
• Filed: 09/26/2008
• Published: 04/02/2009
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: LS9, Inc.
• Inventors: del Cardayre, Stephen B., Bhatia, Monica, Cockrem, Michael C.M., Sanchez-Riera, Fernando A.
• Granted Patent: US 8,313,934 B2
• Time in Patent Office: Days
• US Class Current: 44/307
• CPC Class Codes: C12P 7/649 Biodiesel, i.e. fatty acid alkyl esters
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

There are provided bioproducts and methods of improving production of the bioproducts from engineered microbial cells, the methods comprising: providing a fermentation broth comprising a crude carbon source; inoculating said fermentation broth with said microbial cells; and incubating the inoculated fermentation broth; wherein said bioproduct is a hydrophobic solvent immiscible with said fermentation broth, and wherein a toxic side product present in said crude carbon source is soluble in said hydrophobic solvent. Also, provided are kits for practicing the methods of improving production of bioproducts.

Claims

1. A method of improving production of a bioproduct from engineered microbial cells, comprising:

providing a fermentation broth comprising a crude carbon source;

inoculating said fermentation broth with said microbial cells; and

incubating the inoculated fermentation broth;

wherein said bioproduct is a hydrophobic solvent immiscible with said fermentation broth, and wherein a toxic side product present in said crude carbon source is soluble in said hydrophobic solvent.

2. The method of claim 1, further comprising:

pretreating said fermentation broth with an initial volume of a hydrophobic solvent; and

removing said fermentation broth from said hydrophobic solvent.

3. The method of claim 2, wherein the pretreating step comprises:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of at most about 1;

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

4. The method of claim 2, wherein the pretreating step comprises:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of from about 1;

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

5. The method of claim 1, further comprising:

introducing a volume of a hydrophobic solvent to the provided fermentation broth.

6. The method of claim 5, wherein the introduced volume of hydrophobic solvent is at a ratio of at most 1:

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

7. The method of claim 5, wherein the introduced volume of hydrophobic solvent is at a ratio of from about 1:

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

8. The method of any of claims 1-7, wherein said toxic side product comprises at least one of the following:

furfural;

5-hydroxymethyl furfural;

aliphatic acids;

phenolic compounds;

4-hydroxybenzaldehyde;

4-hydroxybenzoic acid;

hydroquinone;

catechol;

4-methyl catechol;

syringaldehyde;

syringic acid;

guaiacol;

vanillic acid;

vanillin;

1-acetyl-9H-pyrido[3,4B]indole;

1-acetylpyrrolidine;

2,3-dihydrobenzofuran;

2,5-dimethyl-4-hydroxy-3(2H)-furnine;

2,5-dimethylpyrazine;

2-hydroxy-3-methyl-2-cyclopenten-1-one;

5,6-dihydro-6-pentyl-2H-pyran-4-one;

benzeneacetic acid;

benzoic acid;

furan carboyxlic acid methylethylester;

hexadecanoic acid;

hexanoic acid;

pentanoic acid;

trimethylpyrazine;

furan;

n-hexanoic-acid;

or 4-carboxybenzaldehyde.

9. The method of any of claims 1-8, wherein said hydrophobic solvent comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

10. The method of any of claims 1-9, wherein said hydrophobic solvent comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14.0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or biodiesel.

11. The method of any of claims 1-10, wherein said bioproduct comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

12. The method of any of claims 1-11, wherein said bioproduct comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or biodiesel.

13. The method of any of claims 1-12, wherein said bioproduct is a biofuel.

14. The method of any of claims 1-13, wherein said hydrophobic solvent and said bioproduct are similar molecules.

15. The method of any of claims 1-14, wherein said crude carbon source is derived from a biomass.

16. The method of claim 15, wherein said crude carbon source comprises hydrolyzed cellulosic, hydrolyzed hemicellulosic material, or molasses.

17. The method of claim 16, wherein said cellulosic or hemicellulosic material comprises wood, grass, forestry waste, agricultural residue, or agroindustrial residue.

18. The method of any of claims 1-17, wherein the engineered microbial cell is engineered to enhance expression of a gene encoding an enzyme selected from the group consisting of thioesterase, acyl-CoA synthase, and ester synthase.

19. The method of claim 18, wherein a gene encoding an acyl-CoA synthase is attenuated or deleted in the engineered microbial cell.

20. The method of any of claims 19, wherein the gene encoding an acyl-CoA synthase is fadE.

21. A method of passively phase-separating a toxic side product from an aqueous phase of a fermentation broth that is a mixture of crude carbon source and microbial cells engineered to produce a bioproduct during fermentation of a culture of said microbial cells, comprising:

providing said fermentation broth comprising a crude carbon source;

inoculating said fermentation broth with said microbial cells to form a fermentation culture; and

incubating said fermentation culture to yield production of said bioproduct;

wherein said bioproduct is a hydrophobic solvent, and wherein said toxic side product present in said crude carbon source is soluble in said hydrophobic solvent.

22. The method of claim 21, further comprising:

pretreating said fermentation broth with an initial volume of hydrophobic solvent to yield a pretreated fermentation broth; and

removing said fermentation broth from said hydrophobic solvent.

23. The method of claim 22, wherein said pretreating step comprises:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of at most about 1;

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

24. The method of claim 22, wherein said pretreating step comprises:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of from about 1;

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

25. The method of claim 21, further comprising

introducing a volume of a hydrophobic solvent to said fermentation broth.

26. The method of claim 25, wherein the introduced volume of hydrophobic solvent is at a ratio of at most 1:

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

27. The method of claim 25, wherein the introduced volume of hydrophobic solvent is at a ratio of from about 1:

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

28. The method of any of claims 21-27, wherein said toxic side product comprises at least one of the following:

furfural;

5-hydroxymethyl furfural;

aliphatic acids;

phenolic compounds;

4-hydroxybenzaldehyde;

4-hydroxybenzoic acid;

hydroquinone;

catechol;

4-methyl catechol;

syringaldehyde;

syringic acid;

guaiacol;

vanillic acid;

vanillin;

1-acetyl-9H-pyrido[3,4B]indole;

1-acetylpyrrolidine;

2,3-dihydrobenzofuran;

2,5-dimethyl-4-hydroxy-3(2H)-furnine;

2,5-dimethylpyrazine;

2-hydroxy-3-methyl-2-cyclopenten-1-one;

5,6-dihydro-6-pentyl-2H-pyran-4-one;

benzeneacetic acid;

benzoic acid;

furan carboyxlic acid methylethylester;

hexadecanoic acid;

hexanoic acid;

pentanoic acid;

trimethylpyrazine;

furan;

n-hexanoic-acid;

or 4-carboxybenzaldehyde.

29. The method of any of claim 21-27, wherein said hydrophobic solvent comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

30. The method of any of claims 21-27, wherein said hydrophobic solvent comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

0 ester, C2;

C18;

1 ester, or biodiesel.

31. The method of any of claims 21-30, wherein said bioproduct comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

32. The method of any of claims 21-31, wherein said bioproduct comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or biodiesel.

33. The method of any of claims 21-32, wherein said bioproduct is a biofuel.

34. The method of any of claims 21-33, wherein said hydrophobic solvent and said bioproduct are similar molecules.

35. The method of any of claims 21-34, wherein said crude carbon source is derived from a biomass.

36. The method of claim 35, wherein said crude carbon source comprises hydrolyzed cellulosic, hydrolyzed hemicellulosic material, or molasses.

37. The method of claim 36, wherein said cellulosic or hemicellulosic material comprises wood, grass, forestry waste, agricultural residue, or agroindustrial residue.

38. The method of any of claims 21-37, wherein the engineered microbial cell is engineered to enhance expression of a gene encoding an enzyme selected from the group consisting of thioesterase, acyl-CoA synthase, and ester synthase.

39. The method of claim 38, wherein a gene encoding an acyl-CoA synthase is attenuated or deleted in the engineered microbial cell.

40. The method of any of claims 39, wherein the gene encoding an acyl-CoA synthase is fadE.

41. A kit for producing bioproducts from bioengineered cells, comprising:

a sample of bioengineered cells that are bioengineered to produce a bioproduct from a carbon source;

a fermentation broth comprising a crude carbon source derived from conversion of a biomass;

a hydrophobic solvent immiscible with said fermentation broth and capable of extracting a toxic side product formed during conversion of said biomass; and

instructions for a fermentation process using said sample of bioengineered cells, fermentation broth, and hydrophobic solvent.

42. The kit of claim 41, further comprising equipment for growing a culture of the bioengineered cells.

43. The kit of any of claims 41-42, wherein said hydrophobic solvent and said fermentation broth are of volumes resulting in ratios of at most 1:

1 (vol;

vol) hydrophobic solvent;

fermentation broth.

44. The kit of any of claims 41-43, wherein said hydrophobic solvent comprises alkanes, esters olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

45. The kit of any of claims 41-44, wherein said hydrophobic solvent comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or commercial biodiesel.

46. The kit of any of claims 41-45, wherein said bioproduct comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

47. The kit of any of claims 41-46, wherein said bioproduct comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or biodiesel.

48. The kit of any of claims 41-47, wherein said bioproduct is a biofuel.

49. The kit of any of claims 41-48, wherein said hydrophobic solvent and said bioproduct are similar molecules.

50. The kit of any of claims 41-49, wherein the crude carbon source is derived from a biomass.

51. The kit of claim 50, wherein said crude carbon source comprises hydrolyzed cellulosic, hydrolyzed hemicellulosic material, or molasses.

52. The kit of claim 51, wherein said cellulosic or hemicellulosic material comprises wood, grass, forestry waste, agricultural residue, or agroindustrial residue.

53. The kit of any of claims 41-52, wherein the engineered microbial cell is engineered to enhance expression of a gene encoding an enzyme selected from the group consisting of thioesterase, acyl-CoA synthase, and ester synthase.

54. The kit of claim 53, wherein a gene encoding an acyl-CoA synthase is attenuated or deleted in the engineered microbial cell.

55. The kit of any of claims 54, wherein the gene encoding an acyl-CoA synthase is fadE.

56. A continuous fermentation process for the production of a bioproduct from engineered microbial cells, comprising:

providing a culture of said microbial cells;

introducing a continuous flow of volume of a fermentation broth comprising a crude carbon source to said culture;

fermenting said culture with said fermentation broth; and

removing a continuous flow of volume of said fermentation broth from said culture;

wherein said bioproduct is a hydrophobic solvent that is immiscible with said fermentation broth, and wherein a toxic side product present in said crude carbon source is soluble in said hydrophobic solvent.

57. The continuous fermentation process of claim 56, further comprising:

pretreating said fermentation broth with an initial volume of a hydrophobic solvent; and

removing said fermentation broth from said hydrophobic solvent.

58. The continuous fermentation process of claim 57, comprising:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of at most about 1;

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

59. The continuous fermentation process of claim 58, comprising:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of from about 1;

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

60. The continuous fermentation process of claim 56, further comprising

introducing a volume of a hydrophobic solvent to said fermentation broth.

61. The continuous fermentation process of claim 60, wherein the introduced volume of hydrophobic solvent is at a ratio of at most 1:

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

62. The continuous fermentation process of claim 60, wherein the introduced volume of hydrophobic solvent is at a ratio of from about 1:

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

63. The continuous fermentation process of any of claims 56-62, wherein said toxic side product comprises at least one of the following:

furfural;

5-hydroxymethyl furfural;

aliphatic acids;

phenolic compounds;

4-hydroxybenzaldehyde;

4-hydroxybenzoic acid;

hydroquinone;

catechol;

4-methyl catechol;

syringaldehyde;

syringic acid;

guaiacol;

vanillic acid;

vanillin;

1-acetyl-9H-pyrido[3,4B]indole;

1-acetylpyrrolidine;

2,3-dihydrobenzofuran;

2,5-dimethyl-4-hydroxy-3(2H)-furnine;

2,5-dimethylpyrazine;

2-hydroxy-3-methyl-2-cyclopenten-1-one;

5,6-dihydro-6-pentyl-2H-pyran-4-one;

benzeneacetic acid;

benzoic acid;

furan carboyxlic acid methylethylester;

hexadecanoic acid;

hexanoic acid;

pentanoic acid;

trimethylpyrazine;

furan;

n-hexanoic-acid;

or 4-carboxybenzaldehyde.

64. The continuous fermentation process of any of claims 56-63, wherein said hydrophobic solvent comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

65. The continuous fermentation process of any of claims 56-64, wherein said hydrophobic solvent comprises methyl laureate, hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or commercial biodiesel.

66. The continuous fermentation process of any of claims 56-65, wherein said bioproduct comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

67. The continuous fermentation process of any of claims 56-66, wherein said bioproduct comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or biodiesel.

68. The continuous fermentation process of any of claims 56-67, wherein said bioproduct is a biofuel.

69. The continuous fermentation process of any of claims 56-68, wherein said hydrophobic solvent and said bioproduct are a similar molecule.

70. The continuous fermentation process of any of claims 56-69, wherein said crude carbon source is derived from a biomass.

71. The continuous fermentation process of claim 70, wherein said crude carbon source comprises hydrolyzed cellulosic, hydrolyzed hemicellulosic material, or molasses.

72. The continuous fermentation process of claim 71, wherein said cellulosic or hemicellulosic material comprises wood, grass, forestry waste, agricultural residue, or agroindustrial residue.

73. The continuous fermentation process of any of claims 56-72, wherein the engineered microbial cell is engineered to enhance expression of a gene encoding an enzyme selected from the group consisting of thioesterase, acyl-CoA synthase, and ester synthase.

74. The continuous fermentation process of claim 73, wherein a gene encoding an acyl-CoA synthase is attenuated or deleted in the engineered microbial cell.

75. The continuous fermentation process of any of claims 74, wherein the gene encoding an acyl-CoA synthase is fadE.

76. A bioproduct produced by an engineered microbial cell by a process comprising:

preparing a fermentation broth comprising a crude carbon source;

inoculating said fermentation broth with said microbial cells;

incubating the inoculated fermentation broth;

recovering said bioproduct from the incubated fermentation broth;

wherein said bioproduct is a hydrophobic solvent immiscible with said fermentation broth, and wherein a toxic side product present in said crude carbon source is soluble in said hydrophobic solvent.

77. The bioproduct produced by the process of claim 76, said process further comprising:

pretreating said fermentation broth with an initial volume of a hydrophobic solvent; and

removing said fermentation broth from said hydrophobic solvent.

78. The bioproduct produced by the process of claim 77, wherein the pretreating step comprises:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of at most about 1;

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

79. The bioproduct produced by the process of claim 78, wherein the pretreating step comprises:

mixing said initial volume of hydrophobic solvent with said fermentation broth at a ratio of from about 1;

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

80. The bioproduct produced by the process of claim 76, wherein the process further comprises:

introducing a volume of a hydrophobic solvent to the inoculated fermentation broth.

81. The bioproduct produced by the process of claim 80, wherein the introduced volume of hydrophobic solvent is at a ratio of at most 1:

1 (volume;

volume) hydrophobic solvent;

fermentation broth.

82. The bioproduct produced by the process of claim 80, wherein the introduced volume of hydrophobic solvent is at a ratio of from about 1:

3 to about 1;

100 (volume;

volume) hydrophobic solvent;

fermentation broth.

83. The bioproduct produced by the process of any of claims 76-82, wherein the step of recovering comprises:

coalescing said bioproduct in said fermentation broth.

84. The bioproduct produced by the process of any of claims 76-83, said process further comprising;

purifying the recovered bioproduct.

85. The bioproduct produced by the process of claim 84, wherein said purifying step comprises:

centrifuging, filtrating, or solvent extracting.

86. The bioproduct produced by the process of any of claims 84-85, said process further comprising:

polishing said purified bioproduct.

87. The bioproduct produced by the process of claim 86, wherein said polishing step comprises:

deionizing, adsorbing, decolizing, or drying.

88. The bioproduct produced by the process of any of claims 76-87, wherein said toxic side product comprises at least one of the following:

furfural, 5-hydroxymethyl furfural;

aliphatic acids;

phenolic compounds;

4-hydroxybenzaldehyde;

4-hydroxybenzoic acid;

hydroquinone;

catechol;

4-methyl catechol;

syringaldehyde;

syringic acid;

guaiacol;

vanillic acid;

vanillin;

1-acetyl-9H-pyrido[3,4B]indole;

1-acetylpyrrolidine;

2,3-dihydrobenzofuran;

2,5-dimethyl-4-hydroxy-3(2H)-furnine;

2,5-dimethylpyrazine;

2-hydroxy-3-methyl-2-cyclopenten-1-one;

5,6-dihydro-6-pentyl-2H-pyran-4-one;

benzeneacetic acid;

benzoic acid;

furan carboyxlic acid methylethylester;

hexadecanoic acid;

hexanoic acid;

pentanoic acid;

trimethylpyrazine;

furan;

n-hexanoic-acid;

or 4-carboxybenzaldehyde.

89. The bioproduct produced by the process of any of claims 76-88, wherein said hydrophobic solvent comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

90. The bioproduct produced by the process of any of claims 76-89, wherein said hydrophobic solvent comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or commercial biodiesel.

91. The bioproduct produced by the process of any of claims 76-90, wherein said bioproduct comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

92. The bioproduct produced by the process of any of claims 76-91, wherein said bioproduct comprises hexadecane, eicosadecene, C1C12:

0 ester, C1C12;

1 ester, C1C14;

0 ester, C1C14;

1 ester, C1C16;

0 ester, C1C16;

1 ester, C1C18;

1 ester, C2C12;

0 ester, C2C12;

1 ester, C2C14;

0 ester, C2;

C14;

1 ester, C2C16;

0 ester, C2C16;

1 ester, C2;

C18;

1 ester, or biodiesel.

93. The bioproduct produced by the process of any of claims 76-92, wherein said bioproduct is a biofuel.

94. The bioproduct produced by the process of any of claims 76-93, wherein said hydrophobic solvent and said bioproduct are similar molecules.

95. The bioproduct produced by the process of any of claims 76-94, wherein said crude carbon source is derived from a biomass.

96. The bioproduct produced by the process of claim 95, wherein said crude carbon source comprises hydrolyzed cellulosic, hydrolyzed hemicellulosic material, or molasses.

97. The bioproduct produced by the process of claim 96, wherein said cellulosic or hemicellulosic material comprises wood, grass, forestry waste, agricultural residue, or agroindustrial residue.

98. A vehicle powered by energy from combustion of a bioproduct produced by an engineered microbial cell by a process comprising:

preparing a fermentation broth comprising a crude carbon source;

inoculating said fermentation broth with said microbial cells;

incubating the inoculated fermentation broth;

recovering said bioproduct from the incubated fermentation broth;

wherein said bioproduct is a hydrophobic solvent immiscible with said fermentation broth, and wherein a toxic side product present in said crude carbon source is soluble in said hydrophobic solvent.

99. The vehicle of claim 98, said process further comprising:

pretreating said fermentation broth with an initial volume of a hydrophobic solvent; and

removing said fermentation broth from said hydrophobic solvent.

100. The method of any of claims 98-99, wherein the engineered microbial cell is engineered to enhance expression of a gene encoding an enzyme selected from the group consisting of thioesterase, acyl-CoA synthase, and ester synthase.

101. The method of claim 100, wherein a gene encoding an acyl-CoA synthase is attenuated or deleted in the engineered microbial cell.

102. The method of any of claims 101, wherein the gene encoding an acyl-CoA synthase is fadE.