Compositions and methods for producing isoprene

• US 9,909,144 B2
• Filed: 01/04/2016
• Issued: 03/06/2018
• 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:
  US14/987,681
• Publication Number:
  US 20160281113A1
• Time in Patent Office: 792 Days
• Field of Search: None
• 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 cells capable of producing isoprene, the cells comprising (a) heterologous nucleic acid encoding an isoprene synthase polypeptide and (b) two or more heterologous mevalonate (MVA) pathway polypeptides and 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.

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

4. The cells of claim 3, wherein the plant isoprene synthase polypeptide is a poplar isoprene synthase polypeptide or a kudzu isoprene synthase polypeptide.

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

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

(a) an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide or (b) a 1-Deoxyxylulose-5-phosphate synthase (DXS) polypeptide.

7. The cells of claim 1, wherein at least one of the nucleic acids encoding one or more MVA pathway polypeptides is a heterologous nucleic acid or a copy of an endogenous nucleic acid.

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

9. The cells of claim 1, wherein at least one of the nucleic acids encoding the isoprene synthase polypeptide and two or more heterologous mevalonate (MVA) pathway polypeptides is over-expressed.

10. The cells of claim 9, wherein the over-expressed nucleic acid is cloned into a multicopy plasmid.

11. The cells of claim 9, wherein the over-expressed nucleic acid is placed under an inducible promoter or a constitutive promoter.

12. The cells of claim 1, wherein the cells are gram-positive bacterial cells or gram-negative bacterial cells.

13. The cells of claim 12, wherein the 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., and P. alcaligenes cells.

14. The cells of claim 1, wherein the cells are fungal cells.

15. The cells of claim 14, wherein the fungal cells are Aspergillus, yeast, Trichoderma, or Yarrowia cells.

16. The cells of claim 15, where the yeast cells are Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp. cells.

17. The cells of claim 14, where the fungal 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.

18. The cells of claim 1, wherein the cells are algal cells.

19. The cells of claim 18, wherein the algal cells are selected from the group consisting of:

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

20. The cells of claim 1, wherein the cells produce greater than about 400 nmole/g_wcm/hr of isoprene.

21. The cells of claim 1, wherein the cells convert more than about 0.002 molar percent of the carbon that the cells consume from a cell culture medium into isoprene.

22. The cells of claim 1, wherein the cells have an average volumetric productivity of isoprene greater than about 0.1 mg/L_broth/hr.

23. A method for producing isoprene, the method comprising (a) culturing the recombinant cells of claim 1 under suitable conditions for the production of isoprene;

and (b) producing isoprene.

24. The method of claim 23, further comprising (c) recovering the isoprene.