Compositions and methods for producing isoprene

• US 8,709,785 B2
• Filed: 08/30/2012
• Issued: 04/29/2014
• Est. Priority Date: 12/13/2007
• 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., Feher, Frank J., La Duca, Richard, McAuliffe, Joseph C., Miasnikov, Andrei, Peres, Caroline M., Puhala, Aaron S., Sanford, Karl J., Valle, Fernando, Whited, Gregory M.
• Primary Examiner(s): CHOWDHURY, IQBAL HOSSAIN
• Application Number:
  US13/600,109
• Publication Number:
  US 20130071908A1
• Time in Patent Office: 607 Days
• Field of Search: None
• US Class Current: 435/254.2
• 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. Recombinant fungal cells capable of producing isoprene, the cells 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.

2. The cells of claim 1, wherein the heterologous nucleic acid encoding an isoprene synthase polypeptide is operably linked to a promoter, and wherein the production of isoprene by the cells is greater than about 400 nmole/g_wcm/hr.

3. The cells of claim 1, wherein the heterologous nucleic acid encoding an isoprene synthase polypeptide 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.

4. The cells of claim 1, wherein the heterologous nucleic acid encoding an isoprene synthase polypeptide 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.

5. The cells of claim 1, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

6. The cells of claim 5, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

7. The cells of claim 5, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

8. The cells of claim 1, wherein the heterologous nucleic acid encoding an isoprene synthase polypeptide is in a vector.

9. The cells of claim 1, wherein heterologous nucleic acid encoding an isoprene synthase polypeptide is integrated into a chromosome of the cells.

10. The cells of claim 1, further comprising nucleic acids encoding:

(a) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide, and (b) at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or one or more mevalonate (MVA) pathway polypeptides.

11. The cells of claim 10, wherein the nucleic acid encoding an IDI polypeptide of (a) is a heterologous nucleic acid encoding an IDI polypeptide.

12. The cells of claim 10, wherein the nucleic acid encoding an IDI polypeptide of (a) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

13. The cells of claim 10, wherein the nucleic acid encoding a DXS polypeptide of (b) is a heterologous nucleic acid encoding a DXS polypeptide.

14. The cells of claim 10, wherein the nucleic acid encoding a DXS polypeptide of (b) is a copy of an endogenous nucleic acid encoding a DXS polypeptide.

15. The cells of claim 10, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides of (b) is a heterologous nucleic acid.

16. The cells of claim 10, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides of (b) is a copy of an endogenous nucleic acid.

17. The cells of claim 10, wherein the cells comprise two or more polypeptides of the MVA pathway.

18. The cells of claim 10, wherein the cells comprise three or more polypeptides of the MVA pathway.

19. The cells of claim 10, wherein the cells comprise four or more polypeptides of the MVA pathway.

20. The cells of claim 10, wherein the cells comprise polypeptides of the entire MVA pathway.

21. The cells of claim 10, wherein at least one of the nucleic acids encoding a polypeptide of (a) and (b) is in a vector.

22. The cells of claim 10, wherein at least one of the nucleic acids encoding a polypeptide of (a) and (b) is integrated into a chromosome of the cells.

23. The cells of claim 10, wherein at least one of the nucleic acids encoding a polypeptide of (a) and (b) is over-expressed.

24. The cells of claim 23, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

25. The cells of claim 23, wherein the over-expressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

26. The cells of claim 1, wherein the cells are Aspergillus, yeast, or Trichoderma cells.

27. The cells of claim 26, where the yeast cells are Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Yarrowia sp., or Candida sp. cells.

28. The cells of claim 1, where the cells are selected from the group consisting of A. oryzae, A. niger, S. cerevisiae, S. pombe, T. reesei, H. insolens, H. lanuginose, H. grisea, C. lucknowense, A. oryzae, A. niger, A sojae, A. japonicus, A. nidulans, A. aculeatus, A. awamori, F. roseum, F. graminum F. cerealis, F. oxysporuim, F. venenatum, N. crassa, M. miehei, T. viride, F. oxysporum, and F. solan cells.