COMPOSITIONS AND METHODS FOR PRODUCING ISOPRENE

• US 20090203102A1
• Filed: 12/15/2008
• Published: 08/13/2009
• Est. Priority Date: 12/13/2007
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Goodyear Tire & Rubber Company
• Inventors: Valle, Fernando, Fioresi, Carol, Chotani, Gopal K., Sanford, Karl J., Mcauliffe, Joseph C., Miasnikov, Andrei, Cervin, Marguerite A., Aldor, Ilana S., Feher, Frank J., Puhala, Aaron S., Whited, Gregory M.
• Granted Patent: US 8,288,148 B2
• Time in Patent Office: Days
• US Class Current: 435/167
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/1229 Phosphotransferases with a phosphate group as acceptor (2.7.4)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12Y 203/01009 Acetyl-CoA C-acetyltransferase (2.3.1.9)
• CPC Class Codes: C12Y 207/01036 Mevalonate kinase (2.7.1.36)
• CPC Class Codes: C12Y 401/01033 Diphosphomevalonate decarboxylase (4.1.1.33), i.e. mevalonate-pyrophosphate decarboxylase
• CPC Class Codes: C12Y 402/03027 Isoprene synthase (4.2.3.27)
• CPC Class Codes: C12Y 503/03002 Isopentenyl-diphosphate DELTA-isomerase (5.3.3.2)
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

The invention features methods for producing isoprene from cultured cells. The invention also provides compositions that include these cultured cells.

Claims

1. Cells in culture that produce greater than about 400 nmole/g_wcm/hr of isoprene.

8. The cells of claim 1, wherein the cells produce between about 400 nmole/g_wcm/hr to about 2.0×

10⁵ nmole/g_wcm/hr of isoprene.

10. The cells of claim 1, further comprising a heterologous nucleic acid encoding an MVA pathway polypeptide.

2. Cells in culture that have an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr.

3. Cells in culture that convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene.

4. Cells in culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the cells (i) produce greater than about 400 nmole/g_wcm/hr of isoprene, (ii) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, or (iii) have an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr.

9. The cells of claim 4, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

5. Cells in culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the production of isoprene by the cells is greater than about 400 nmole/g_wcm/hr.

6. Cells in culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells have an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr.

7. Cells in culture comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium is converted into isoprene.

11. A method for producing isoprene, the method comprising culturing cells under conditions sufficient to produce greater than about 400 nmole/g_wcm/hr of isoprene.

18. The method of claim 11, further comprising recovering the isoprene produced by the cells.

20. The method of claim 11, wherein the cells further comprise a heterologous nucleic acid encoding an MVA pathway polypeptide.

12. A method for producing isoprene, the method comprising culturing cells under conditions resulting in an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr.

13. A method for producing isoprene, the method comprising culturing cells under conditions sufficient to convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene.

14. A method for producing isoprene, the method comprising culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein production of isoprene by the cells is greater than about 400 nmole/g_wcm/hr.

19. The method of claim 14, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

15. A method for producing isoprene, the method comprising culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein the cells have an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr.

16. A method for producing isoprene, the method comprising culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide, wherein the heterologous nucleic acid is operably linked to a promoter, and wherein more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium is converted into isoprene.

17. A method of producing isoprene, the method comprising

(a) culturing cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene, wherein the cells (i) produce greater than about 400 nmole/g_wcm/hr of isoprene, (ii) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene, or (iii) have an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr of isoprene, and

(b) producing isoprene.