Compositions and methods for producing isoprene free of C5 hydrocarbons under decoupling conditions and/or safe operating ranges

• US 8,420,360 B2
• Filed: 07/01/2009
• Issued: 04/16/2013
• Est. Priority Date: 07/02/2008
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company, Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Original Assignee: Goodyear Tire & Rubber Company, Danisco US, Inc. (International Flavors & Fragrances Incorporated)
• Inventors: Cervin, Marguerite A., Chotani, Gopal K., La Duca, Richard, McAuliffe, Joseph C., Miller, Michael C., Sanford, Karl J., Whited, Gregory M., Sabo, Timothy A., Webster, Erin L., Calabria, Anthony R.
• Primary Examiner(s): Kosson, Rosanne
• Application Number:
  US12/496,573
• Publication Number:
  US 20100048964A1
• Time in Patent Office: 1,385 Days
• Field of Search: 435/167, 585/16
• US Class Current: 435/167
• 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. A method of producing isoprene, the method comprising

(a) culturing recombinant cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under suitable culture conditions for the production of isoprene in a gas phase;

and

(b) producing isoprene in the gas phase, wherein the gas phase comprises a nonflammable concentration of isoprene and less than or about 9.5% (volume) oxygen, and wherein the cells produce greater than about 400 nmole/g_wcm/hr of isoprene.

2. The method of claim 1, wherein the concentration of isoprene in the gas phase is less than the lower flammability limit (LFL) of isoprene.

3. The method of claim 2, wherein the LFL of isoprene is from about 1.5 vol. % to about 2.0 vol. %.

4. The method of claim 1, wherein the concentration of isoprene in the gas phase is greater than the upper flammability limit (UFL) of isoprene.

5. The method of claim 4, wherein the UFL of isoprene is from about 2.0 vol. % to about 12.0 vol. %.

6. The method of claim 1, wherein isoprene is produced in the gas phase at a temperature between about 25°

C. to about 55°

C.

7. The method of claim 1, wherein isoprene is produced in the gas phase at a pressure between about 1 atmosphere and 3 atmospheres.

8. The method of claim 1, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

9. The method of claim 1, wherein the cells are bacterial cells.

10. The method of claim 9, wherein the bacterial cells are selected from the group consisting of Escherichia coli, Pantoae citrea, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis, Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus, Pseudomonas sp., and Pseudomonas alcaligenes cells.

11. The method of claim 1, wherein the cells are fungal cells.

12. The method of claim 11, wherein the fungal cells are selected from the group consisting of Aspergillus sp., yeast, Trichoderma sp., Yarrowia sp., Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Candida sp., Aspergillus oryzae, Aspergillus niger, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma reesei, H. insolens, H. lanuginose, H. grisea, Candida lucknowense, Aspergillus sojae, Aspergillus japonicus, Aspergillus nidulans, Aspergillus aculeatus, Aspergillus awamori, Fusarium roseum, Fusarium graminum, Fusarium cerealis, Fusarium oxysporuim, F. venenatum, Neurospora crassa, Mucor miehei, Trichoderma viride, Fusarium oxysporum, and Fusarium solani cells.

13. The method of claim 1, wherein the cells are algal cells.

14. The method of claim 13, wherein the algal cells are selected from the group consisting of green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, and dinoflagellates.

15. The method of claim 1, wherein the cells further comprise a nucleic acid encoding an isopentyl-diphosphate delta-isomerase (IDI) polypeptide.

16. The method of claim 15, wherein the nucleic acid encoding the IDI polypeptide is a heterologous nucleic acid.

17. The method of claim 15, wherein the IDI polypeptide is a yeast IDI polypeptide.

18. The method of claim 15, wherein the nucleic acid encoding the IDI polypeptide is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

19. The method of claim 1, wherein the cells further comprise a heterologous nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase (DXS) polypeptide.

20. The method of claim 1, wherein the cells further comprise a copy of an endogenous nucleic acid encoding a DXS polypeptide.

21. The method of claim 1, wherein the cells further comprise one or more heterologous nucleic acids encoding one or more mevalonate (MVA) pathway polypeptides.

22. The method of claim 1, wherein the cells further comprise one or more copies of endogenous nucleic acids encoding one or more MVA pathway polypeptides.

23. The method of claim 1, wherein the cells further comprise one or more nucleic acids encoding the polypeptides of the entire MVA pathway.

24. The method of claim 1, wherein the cells further comprise:

(a) a nucleic acid encoding an isopentyl-diphosphate delta-isomerase (IDI) polypeptide; and

(b) (i) a nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or (ii) one or more nucleic acids encoding one or more mevalonate (MVA) pathway polypeptides.

25. The method of claim 24, wherein the nucleic acid encoding a DXS polypeptide is a heterologous nucleic acid.

26. The method of claim 24, wherein the nucleic acid encoding a DXS polypeptide is a copy of an endogenous nucleic acid.

27. The method of claim 24, wherein said one or more nucleic acids encoding one or more MVA pathway polypeptides are heterologous nucleic acids.

28. The method of claim 24, wherein said one or more nucleic acids encoding one or more MVA pathway polypeptides are endogenous nucleic acids.