Compositions and methods for improved isoprene production using two types of ISPG enzymes

• US 8,685,702 B2
• Filed: 12/22/2011
• Issued: 04/01/2014
• Est. Priority Date: 12/22/2010
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Muir, Rachel E., Weyler, Walter
• Primary Examiner(s): CHOWDHURY, IQBAL HOSSAIN
• Application Number:
  US13/335,768
• Publication Number:
  US 20120164711A1
• Time in Patent Office: 831 Days
• Field of Search: None
• US Class Current: 435/252.3
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/0093 acting on CH or CH2 groups (1.17)
• CPC Class Codes: C12N 9/1022 transferring aldehyde or ketonic groups (2.2)
• 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

Abstract

The invention provides for compositions and methods for producing isoprene by using recombinantly engineered cells that utilize a system of dual IspG enzymes in addition to isoprene synthase.

Claims

1. A recombinant cell capable of producing isoprene, the cell comprising:

(a) a nucleic acid encoding a first 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate synthase (IspG) polypeptide of a first species and a nucleic acid encoding a second IspG polypeptide of a second species, wherein the second species differs from the first species;

(b) a nucleic acid encoding at least one 1-deoxyxylulose-5-phosphate (DXP) pathway enzyme, and (c) a nucleic acid encoding an isoprene synthase polypeptide, wherein the cell produces isoprene at a greater titer as compared to a cell comprising a nucleic acid encoding an IspG polypeptide of a single species.

2. The recombinant cell of claim 1, wherein the first IspG polypeptide is a T. elongatus IspG polypeptide.

3. The recombinant cell of claim 1, wherein the second IspG polypeptide is an E. coli IspG polypeptide.

4. The recombinant cell of claim 1, further comprising a nucleic acid encoding an iron-sulfur cluster-interacting redox polypeptide.

5. The recombinant cell of claim 4, wherein the iron-sulfur cluster-interacting redox polypeptide is selected from ferredoxin and flavodoxin.

6. The recombinant cell of claim 1, further comprising a nucleic acid encoding a 1-deoxyxylulose-5-phosphate (DXP) pathway associated polypeptide.

7. The recombinant cell of claim 6, wherein the 1-deoxyxylulose-5-phosphate (DXP) pathway associated polypeptide is a chaperone protein.

8. The recombinant cell of claim 1, wherein the cell further comprises at least one heterologous nucleic acid encoding an isopentenyl-diphosphate delta-isomerase (IDI) polypeptide or at least one copy of an endogenous nucleic acid encoding an IDI polypeptide.

9. The recombinant cell of claim 1, wherein the cell further comprises at least one heterologous nucleic acid encoding a mevalonate (MVA) pathway polypeptide or at least one copy of an endogenous nucleic acid encoding a MVA pathway polypeptide.

10. The recombinant cell of claim 8, wherein the cell further comprises at least one heterologous nucleic acid encoding a mevalonate (MVA) pathway polypeptide or at least one copy of an endogenous nucleic acid encoding a MVA pathway polypeptide.

11. The recombinant cell of claim 1, wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

12. The recombinant cell of claim 11, wherein the isoprene synthase polypeptide is a polypeptide from Pueraria or Populus or a hybrid, Populus alba ×

Populus tremula.

13. The recombinant cell of claim 11, wherein the isoprene synthase polypeptide is selected from the group consisting of Pueraria montana or Pueraria lobata, Populus tremuloides, Populus alba, Populus nigra, and Populus trichocarpa.

14. The recombinant cell of claim 11, wherein the plant isoprene synthase polypeptide is a kudzu isoprene synthase polypeptide.

15. The recombinant cell of claim 1, wherein the cell is a bacterial, algal, fungal or yeast cell.

16. The recombinant cell of claim 15, wherein the cell is a bacterial cell.

17. The bacterial cell of claim 16, wherein the bacterial cell is a gram-positive bacterial cell or gram-negative bacterial cell.

18. The bacterial cell of claim 17, wherein the bacterial cell is 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.

19. The recombinant cell of claim 1, wherein the additional 1-deoxyxylulose-5-phosphate (DXP) pathway enzyme is selected from the group of:

1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (MCT), 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase, (CMK), 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (MCS), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR/IspH), and isopentenyl-diphosphate delta isomerase (IDI).

20. The recombinant cell of claim 19, wherein the additional 1-deoxyxylulose-5-phosphate (DXP) pathway enzyme is selected from the group of:

1-deoxyxylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR/IspH), and isopentenyl-diphosphate delta isomerase (IDI).