Compositions and methods for producing isoprene

• US 9,260,727 B2
• Filed: 08/30/2012
• Issued: 02/16/2016
• Est. Priority Date: 12/13/2007
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (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:
  US13/600,132
• Publication Number:
  US 20130078699A1
• Time in Patent Office: 1,265 Days
• Field of Search: None
• US Class Current: 1/1
• 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. A method of producing isoprene, the method comprising:

(a) culturing recombinant cells under suitable culture conditions for the production of isoprene, wherein the cells comprise nucleic acids encoding

(i) a heterologous isoprene synthase polypeptide,

(ii) an isopentenyl-diphosphate delta-isomerase (DI) polypeptide, and

(iii) at least one of a 1-Deoxyxylulose-5-phosphate synthase (DXS)polypeptide and/or one or more mevalonate (MVA) pathway polypeptides and wherein the cells

(1) produce greater than about 400 nmole/gwcm/hr of isoprene,

(2) convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene,

(3) have an average volumetric productivity of isoprene greaterthan about 0.1mg/Lbroth/hr of isoprene, or

(4) produce at least 0.0037 grams of isoprene per gram of dry cell mass (gdcm); and

(b) producing isoprene.

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

3. The method of claim 1, wherein the cells further comprise an endogenous nucleic acid encoding an isoprene synthase polypeptide.

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

5. The method of claim 4, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide.

6. The method of claim 4, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

7. The method of claim 1, wherein the nucleic acid encoding an IDI polypeptide of (a)(ii) is a heterologous nucleic acid encoding an IDI polypeptide.

8. The method of claim 1, wherein the nucleic acid encoding an IDI polypeptide of (a)(ii) is a copy of an endogenous nucleic acid encoding an IDI polypeptide.

9. The method of claim 1, wherein the nucleic acid encoding a DXS polypeptide of (a)(iii) is a heterologous nucleic acid encoding a DXS polypeptide.

10. The method of claim 1, wherein the nucleic acid encoding a DXS polypeptide of (a)(iii) is a copy of an endogenous nucleic acid encoding a DXS polypeptide.

11. The method of claim 1, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides of (a)(iii) is a heterologous nucleic acid.

12. The method of claim 1, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides of (a)(iii) is a copy of an endogenous nucleic acid.

13. The method of claim 1, wherein the cells comprise two or more polypeptides of the MVA pathway.

14. The method of claim 1, wherein the cells comprise three or more polypeptides of the MVA pathway.

15. The method of claim 1, wherein the cells comprise four or more polypeptides of the MVA pathway.

16. The method of claim 1, wherein the cells comprise polypeptides of the entire MVA pathway.

17. The method of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a)(i)-(a)(iii) is in a vector.

18. The method of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a)(i)-(a)(iii) is integrated into a chromosome of the cells.

19. The method of claim 1, wherein at least one of the nucleic acids encoding a polypeptide of (a)(i)-(a)(iii) is over-expressed.

20. The method of claim 19, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

21. The method of claim 20, wherein the over-expressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

22. The method of claim 1, wherein the cells are fungal, bacterial, plant, or algal cells.

23. The method of claim 22, wherein the cells are fungal cells.

24. The method of claim 23, wherein the cells are Aspergillus, yeast, or Trichoderma, cells.

25. The method of claim 24, wherein the yeast cells are Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Yarrowia sp. or Candida sp. cells.

26. The method cells of claim 23, 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. 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.

27. The method of claim 22, wherein the cells are bacterial cells.

28. The method of claim 27, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

29. The method of claim 27, wherein the bacterial cells are selected from the group consisting of E. coli, P. citrea, B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B.megaterium, B. coagulans, B. circulans, B. lautus, B. thuringiensis, S. albus, S. lividans, S. coelicolor, S. griseus, Pseudomonas sp., Corynebacteria sp., and P. alcaligenes cells.

30. The method of claim 22, wherein the cells are plant cells.

31. The method of claim 30, wherein the plant cells are selected from the group consisting of:

the family Fabaceae, the Faboideae subfamily, kudzu, poplar, Populus alba×

tremula, Populus alba, aspen, Populus tremuloides, and Quercus robur cells.

32. The method of claim 22, wherein the cells are algal cells.

33. The method of claim 32, wherein the algal cells are selected from the group consisting of:

green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, and dinoflagellates.