Systems using cell culture for production of isoprene

• US 8,569,026 B2
• Filed: 09/15/2009
• Issued: 10/29/2013
• Est. Priority Date: 09/15/2008
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Chotani, Gopal K., McAuliffe, Joseph C., Peres, Caroline M., Whited, Gregory M.
• Primary Examiner(s): Kosson, Rosanne
• Application Number:
  US12/560,366
• Publication Number:
  US 20100167371A1
• Time in Patent Office: 1,505 Days
• Field of Search: 435/167, 435/296.1, 435/196
• 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 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 250 M/atm and (b) a solubility in water of less than 100 g/L, the method comprising:

a) culturing cells comprising one or more nucleic acids encoding for an isoprene synthase polypeptide;

b) adding an oxygen-containing gas to the culture at a gas sparging rate between about 0.01 vvm to about 2 vvm, thereby producing the compound; and

c) recovering the compound made in step (b).

2. The method of claim 1, wherein the compound produced is ethylene.

3. The method of claim 1, wherein the compound produced is isoprene.

7. 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 isopentenyl-diphosphate-delta-isomerase (IDI) polypeptide, a mevalonic acid (MVA) pathway enzyme, or a deoxyxylulose-5-phosphate synthase (DXP) pathway enzyme.

8. The method of claim 7 wherein the MVA pathway enzyme is one or more enzymes selected from the group consisting of acetyl-CoA acetyltransferase (AA-CoA thiolase), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), phosphomevalonate decarboxylase (PMDC), and isopentenyl diphosphate isomerase (IDI).

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

Populus tremula, or variants thereof.

10. The method of claim 9, wherein the isoprene synthase polypeptide is selected from the group consisting of Pueraria montana, Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra, Populus trichocarpa, and variants thereof.

11. 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, Corynebacterium cells, Aspergillus cells, or yeast cells.

12. The method of claim 11, 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, Aspergillus niger, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Yarrowia lipolytica.

13. The method of claim 3, wherein the cells produce greater than 400 nmole/gwcm/hour of isoprene and the carbon dioxide evolution rate of the cells is greater than 1×

10<sup>−

18</sup> mmol/L/hour.

4. 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 250 M/atm and (b) a solubility in water of less than 100 g/L, the method comprising:

a) culturing cells comprising one or more nucleic acids encoding for an isoprene synthase polypeptide;

b) adding an oxygen-containing gas to the culture at a gas sparging rate of at least 0.1 vvm, thereby producing the compound; and

c) recovering the compound from the gas phase.

5. The method of claim 4, wherein the compound produced is ethylene.

6. The method of claim 4, wherein the compound is isoprene.

14. The method of claim 4, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an isopentenyl-diphosphate-delta-isomerase (IDI) polypeptide, a mevalonic acid (MVA) pathway enzyme, or a deoxyxylulose-5-phosphate synthase (DXP) pathway enzyme.

15. The method of claim 14 wherein the MVA pathway enzyme is one or more enzymes selected from the group consisting of acetyl-CoA acetyltransferase (AA-CoA thiolase), 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase), 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), mevalonate kinase (MVK), phosphomevalonate kinase (PMK), diphosphomevalonate decarboxylase (MVD), phosphomevalonate decarboxylase (PMDC), and isopentenyl diphosphate isomerase (IDI).

16. The method of claim 4, wherein the isoprene synthase polypeptide is from Pueraria or Populus or the hybrid Populus alba×

Populus tremula, or variants thereof.

17. The method of claim 16, wherein the isoprene synthase polypeptide is selected from the group consisting of Pueraria montana, Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra, Populus trichocarpa, and variants thereof.

18. The method of claim 4, 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, Corynebacterium cells, Aspergillus cells, or yeast cells.

19. The method of claim 18, 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, Aspergillus niger, Saccharomyces cerevisiae, Corynebacterium glutamicum, and Yarrowia lipolytica.

20. The method of claim 6, wherein the cells produce greater than 400 nmole/gwcm/hour of isoprene and the carbon dioxide evolution rate of the cells is greater than 1×

10<sup>−

18</sup> mmol/L/hour.