COMPOSITIONS AND METHODS FOR PRODUCING ISOPRENE FREE OF C5 HYDROCARBONS UNDER DECOUPLING CONDITIONS AND/OR SAFE OPERATING RANGES

• US 20100048964A1
• Filed: 07/01/2009
• Published: 02/25/2010
• Est. Priority Date: 07/02/2008
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company, Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Inventors: CERVIN, Marguerite A., MILLER, Michael C., WEBSTER, Erin L., SABO, Timothy A., MCAULIFFE, Joseph C., CHOTANI, Gopal K., CALABRIA, Anthony R.
• Granted Patent: US 8,420,360 B2
• Time in Patent Office: Days
• US Class Current: 585/16
• CPC Class Codes: C07C 11/18 Isoprene
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/70 Vectors or expression systems specially adapted for E. coli
• CPC Class Codes: C12N 15/74 Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
• CPC Class Codes: C12N 15/75 for Bacillus
• CPC Class Codes: C12N 15/80 for fungi
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12P 5/02 acyclic C12P5/007 takes precedence
• CPC Class Codes: C12Y 402/03027 Isoprene synthase (4.2.3.27)
• 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 wherein the cells in the stationary phase. The invention also provides compositions that include these cultured cells and/or increased amount of isoprene. The invention also provides for systems that include a non-flammable concentration of isoprene in the gas phase. Additionally, the invention provides isoprene compositions, such as compositions with increased amount of isoprene or increased purity.

Claims

1. Cells in culture capable of producing isoprene wherein the cells are in stationary phase and wherein the isoprene production is greater than or about 2-fold more than the amount of isoprene produced during growth phase for the same length of time.

2. The cells of claim 1 wherein the isoprene is produced in the gas phase and

(a) wherein the gas phase comprises greater than or about 9.5% (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit or

(b) the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit, and the cells produce greater than about 400 nmole/g_wcm/hr of isoprene.

6. The cells of claim 1 wherein the cells are grown under limited glucose conditions.

7. The cells of claim 1 wherein the amount of isoprene produced during stationary phase is greater than or about 2, 3, 4, 5, 10, 20, 30, 40, 50, or more times the amount of isoprene produced during the growth phase for the same length of time.

8. The cells of claim 1 further comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide.

9. A composition comprising greater than about 2 mg of isoprene produced by the cells of claim 1 and comprising greater than or about 99.94% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

10. A composition comprising greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 mg of isoprene produced by the cells of claim 1.

11. A composition comprising greater than or about 2, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 g of isoprene (w/w) of the volatile organic fraction from the cells of claim 1.

12. A composition comprising greater than about 2 mg of isoprene produced by the cells of claim 1 and comprising less than or about 0.5 ug/L per compound for any compound in the composition that inhibits the polymerization of isoprene.

13. The composition of claim 12 wherein the composition that inhibits the polymerization of isoprene comprises less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% of one or more C5 hydrocarbons selected from the group consisting of:

1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene, cis-piperylene, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, and cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the composition.

14. The composition of claim 12 wherein the composition that inhibits the polymerization of isoprene has less than or about 0.12, 0.10, 0.08, 0.06, 0.04, 0.02, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005, or 0.00001% of 1,3-cyclopentadiene, cis-1,3-pentadiene, trans-1,3-pentadiene, 1-pentyne, 2-pentyne, 1-pentene, 2-methyl-1-butene, 3-methyl-1-butyne, trans-piperylene, cis-piperylene, pent-4-ene-1-yne, trans-pent-3-ene-1-yne, or cis-pent-3-ene-1-yne by weight compared to the total weight of all C5 hydrocarbons in the composition.

15. A composition comprising greater than about 2 mg of isoprene produced by the cells of claim 1 and has greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the composition.

16. The composition of claim 15 wherein the isoprene is recovered from an off-gas portion.

17. A composition comprising greater than about 2 mg of isoprene produced by the cells of claim 1 and comprising one or more compounds selected from the group consisting of ethanol, acetone, C5 prenyl alcohols, and isoprenoid compounds with 10 or more carbon atoms.

18. A composition comprising greater than about 2 mg of isoprene produced by the cells of claim 1 and comprising one or more second compounds selected from the group consisting of 2-heptanone, 6-methyl-5-hepten-2-one, 2,4,5-trimethylpyridine, 2,3,5-trimethylpyrazine, citronellal, acetaldehyde, methanethiol, methyl acetate, 1-propanol, diacetyl, 2-butanone, 2-methyl-3-buten-2-ol, ethyl acetate, 2-methyl-1-propanol, 3-methyl-1-butanal, 3-methyl-2-butanone, 1-butanol, 2-pentanone, 3-methyl-1-butanol, ethyl isobutyrate, 3-methyl-2-butenal, butyl acetate, 3-methylbutyl acetate, 3-methyl-3-but-1-enyl acetate, 3-methyl-2-but-1-enyl acetate, (E)-3,7-dimethyl-1,3,6-octatriene, (Z)-3,7-dimethyl-1,3,6-octatriene, and 2,3-cycloheptenolpyridine;

wherein the amount of the second compound relative to the amount of the isoprene is greater than or about 0.01% (w/w).

19. A composition comprising (i) a gas phase that comprises isoprene and (ii) cells in culture of claim 1.

20. The composition of claim 19 wherein the composition comprises a closed system, and the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ug/L of isoprene when normalized to 1 mL of 1 OD₆₀₀ cultured for 1 hour.

21. The composition of claim 19 wherein the composition comprises an open system, and the gas phase comprises greater than or about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ug/L of isoprene when sparged at a rate of 1 vvm.

22. The composition of claim 19 wherein the composition comprises a volatile organic fraction of the gas phase comprising greater than or about 99.90, 99.92, 99.94, 99.96, 99.98, or 100% isoprene by weight compared to the total weight of all C5 hydrocarbons in the volatile organic fraction.

23. A method of producing isoprene, the method comprising

(a) culturing cells of claim 1 under suitable culture conditions for the production of isoprene;

wherein the amount of isoprene produced during stationary phase is greater than or about 2-fold more than the amount of isoprene produced during growth phase for the same length of time, and

(b) producing isoprene.

24. The method of claim 23 wherein the cells are cultured under limited glucose conditions.

25. The method of claim 23 wherein the isoprene is recovered from an off-gas portion of the cell culture.

3. Cells in culture that produce greater than about 400 nmole/g_wcm/hr of isoprene wherein the cells are grown under conditions that decouple isoprene production from cell growth.

4. Cells in culture that have an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr wherein the cells are grown under conditions that decouple isoprene production from cell growth.

5. 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 wherein the cells are grown under conditions that decouple isoprene production from cell growth.

26. A system comprising a nonflammable concentration of isoprene in the gas phase wherein the gas phase comprises less than about 9.5% (volume) oxygen or greater than or about 9.5% (volume) oxygen, and the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit.

27. The system of claim 26 wherein the portion of the gas phase other than isoprene comprises between about 10% to about 100% (volume) oxygen.

28. The system of claim 26 wherein the portion of the gas phase other than isoprene comprises between about 0% to about 99% (volume) nitrogen.

29. The system of claim 26 wherein the portion of the gas phase other than isoprene comprises between about 1% to about 50% (volume) CO₂.

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

(a) culturing cells under suitable culture conditions for the production of isoprene, wherein the gas phase comprises greater than or about 9.5% (volume) oxygen, and

(b) producing isoprene, wherein the concentration of isoprene in the gas phase is less than the lower flammability limit or greater than the upper flammability limit, and wherein the cells produce greater than about 400 nmole/g_wcm/hr of isoprene.

31. A method of producing isoprene, the method comprising

(a) culturing cells under suitable culture conditions for the production of isoprene, wherein the gas phase comprises less than or about 9.5% (volume) oxygen, and

(b) producing isoprene, wherein the cells produce greater than about 400 nmole/g_wcm/hr of isoprene.