Reduction of the toxic effect of impurities from raw materials by extractive fermentation

• US 8,313,934 B2
• Filed: 09/26/2008
• Issued: 11/20/2012
• Est. Priority Date: 09/27/2007
• Status: Active Grant

• Current Assignee: Genomatica Incorporated
• Original Assignee: LS9, Inc.
• Inventors: Bhatia, Monica, del Cardayre, Stephen B., Sanchez-Riera, Fernando A., Cockrem, Michael C. M.
• Primary Examiner(s): Ketter, Jim
• Application Number:
  US12/239,562
• Publication Number:
  US 20090084025A1
• Time in Patent Office: 1,516 Days
• Field of Search: None
• US Class Current: 435/135
• 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 producing a bioproduct from engineered microbial cells, comprising:

(a) providing an aqueous fermentation broth comprising a crude carbon source;

(b) inoculating said fermentation broth with microbial cells, engineered to enhance expression of one or more of a gene encoding an enzyme selected from the group consisting of thioesterase, acyl-CoA synthase, and ester synthase, and wherein a gene encoding an acyl-CoA dehydrogenase is attenuated or absent in the engineered microbial cells;

(c) incubating the inoculated fermentation broth;

(d) thereby producing and secreting a bioproduct; and

(e) treating said fermentation broth with a hydrophobic solvent 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

separating 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, wherein said toxic side product comprises at least one compound selected from the group consisting of 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 carboxylic acid methylethylester;

hexadecanoic acid;

hexanoic acid;

pentanoic acid;

trimethylpyrazine;

furan;

n-hexanoic-acid; and

4-carboxybenzaldehyde.

6. The method of claim 1, wherein said hydrophobic solvent comprises alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

7. The method of claim 6, 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.

8. The method of claim 1, wherein said bioproduct comprises one or more of alkanes, esters, olefins, fatty acids, fatty alcohols, aldehydes, or ketones.

9. The method of claim 8, 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.

10. The method of claim 1, wherein said bioproduct is a biofuel.

11. The method of claim 1, wherein said hydrophobic solvent and said bioproduct comprise similar molecules.

12. The method of claim 1, wherein said crude carbon source is derived from biomass.

13. The method of claim 12, wherein said crude carbon source comprises hydrolyzed cellulosic material, hydrolyzed hemicellulosic material, or molasses.

14. The method of claim 13, wherein said cellulosic or hemicellulosic material comprises wood, grass, forestry waste, agricultural residue, or agroindustrial residue.

15. A method of producing a bioproduct from engineered microbial cells, comprising:

(a) providing an aqueous fermentation broth comprising a crude carbon source;

(b) inoculating said fermentation broth with engineered microbial cells wherein the engineered microbial cells are engineered to enhance expression of a gene encoding an enzyme selected from the group consisting of thioesterase, acyl-CoA synthase, and ester synthase and wherein a gene encoding an acyl-CoA dehydrogenase is attenuated or absent in the engineered microbial cell; and

(c) incubating the inoculated fermentation broth,

wherein the engineered microbial cells produce a bioproduct and said bioproduct is a hydrophobic solvent immiscible with said fermentation broth, wherein any toxic side product present in said crude carbon source is soluble in a hydrophobic solvent.

16. The method of claim 15, wherein the gene encoding an acyl-CoA dehydrogenase is fadE.