SYSTEMS USING CELL CULTURE FOR PRODUCTION OF ISOPRENE

• US 20100167371A1
• Filed: 09/15/2009
• Published: 07/01/2010
• Est. Priority Date: 09/15/2008
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company
• Inventors: Chotani, Gopal K., McAuliffe, Joseph C., Peres, Caroline M., Whited, Gregory M.
• Granted Patent: US 8,569,026 B2
• Time in Patent Office: Days
• US Class Current: 435/167
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12P 5/026 Unsaturated compounds, i.e. alkenes, alkynes or allenes
• 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. The invention provides isoprene compositions, such as compositions with increased amount of isoprene or increased purity. Additionally, the invention provides methods of producing isoprene by culturing cells under conditions suitable for isoprene production while maintaining cell viability and/or metabolic activity.

Claims

1. A method of producing isoprene, the method comprising:

a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of isoprene; and

b) producing isoprene, wherein the cells produce greater than about 400 nmole/g_wcm/hour of isoprene, and wherein the carbon dioxide evolution rate of the cells is greater than about 1×

10<sup>−

18</sup> mmol/L/hour.

2. The method of claim 1, further comprising recovering the isoprene.

3. The method of claim 1, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

4. The method of claim 1, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an IDI polypeptide, an MVA pathway enzyme, or a DXP pathway enzyme.

5. The method of claim 4 wherein the MVA pathway enzyme is mevalonate kinase.

6. The method of claim 1, wherein the isoprene synthase polypeptide is from Pueraria or Populus or a hybrid, Populus alba×

Populus tremula.

7. The method of claim 6, wherein the isoprene synthase polypeptide is selected from the group consisting of Pueraria montana or Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra, and Populus trichocarpa.

8. The method of claim 1, wherein the cells are gram-positive bacterial cells, Streptomyces cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, fungal cells, filamentous fungal cells, Trichoderma cells, Aspergillus cells, or yeast cells.

9. The method of claim 8, wherein the cells are selected from the group consisting of Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus, Escherichia coli, Pantoea citrea, Trichoderma reesei, Aspergillus oryzae and Aspergillus niger, Saccharomyces cerevisiae and Yarrowia lipolytica.

10. A method of producing isoprene, the method comprising:

a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of isoprene; and

b) producing isoprene, wherein the liquid phase concentration of isoprene is less than about 1 g/L and the cells produce greater than about 400 nmole/g_wcm/hour of isoprene.

11. The method of claim 10, further comprising recovering the isoprene.

12. The method of claim 10, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

13. The method of claim 10, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an IDI polypeptide, an MVA pathway enzyme, or a DXP pathway enzyme.

14. The method of claim 13 wherein the MVA pathway enzyme is mevalonate kinase.

15. The method of claim 10, wherein the isoprene synthase polypeptide is from Pueraria or Populus or a hybrid, Populus alba×

Populus tremula.

16. The method of claim 10, wherein the cells are gram-positive bacterial cells, Streptomyces cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, fungal cells, filamentous fungal cells, Trichoderma cells, Aspergillus cells, or yeast cells.

17. A method of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry'"'"'s law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L, the method comprising:

a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of the compound, wherein a gas is added at a gas sparging rate between about 0.01 vvm to about 2 vvm; and

b) producing the compound.

18. The method of claim 17, further comprising recovering the compound.

19. The method of claims 17, wherein the compound produced is ethylene.

20. The method of claim 17, wherein the compound produced is isoprene.

21. Cells in culture comprising a nucleic acid encoding an isoprene synthase polypeptide, wherein the cells produce greater than about 400 nmole/g_wcm/hour of isoprene and carbon dioxide evolution rate of the cells is greater than about 1×

10<sup>−

18</sup> mmol/L/hour.

22. A system comprising a reaction chamber with cells of claim 21.

23. A method of producing a compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry'"'"'s law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L, the method comprising:

a) culturing cells comprising one or more nucleic acids encoding for isoprene synthase polypeptide under suitable conditions for production of the compound, wherein the compound has one or more characteristics selected from the group consisting of (a) a Henry'"'"'s law coefficient of less than about 250 M/atm and (b) a solubility in water of less than about 100 g/L;

b) producing the compound;

c) and recovering the compound from the gas phase.

24. The method of claim 23, wherein the compound produced is ethylene.

25. The method of claim 23, wherein the compound is isoprene.