INCREASED ISOPRENE PRODUCTION USING THE ARCHAEAL LOWER MEVALONATE PATHWAY

• US 20100086978A1
• Filed: 09/15/2009
• Published: 04/08/2010
• Est. Priority Date: 09/15/2008
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company
• Inventors: Calabria, Anthony R., Miller, Michael C., Vaviline, Dmitrii V., Beck, Zachary Q.
• Granted Patent: US 8,361,762 B2
• Time in Patent Office: Days
• US Class Current: 435/131
• CPC Class Codes: C12N 9/1205 Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: C12P 9/00 Preparation of organic compounds containing a metal or atom other than H, N, C, O, S or halogen phosphoglycerides, C12P7/6481
• CPC Class Codes: C12Y 207/01036 Mevalonate kinase (2.7.1.36)
• CPC Class Codes: C12Y 402/03027 Isoprene synthase (4.2.3.27)
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

The invention features methods for producing isoprene from cultured cells using a feedback-resistant mevalonate kinase polypeptide, such as an archaeal mevalonate kinase polypeptide. The resulting isoprene compositions may have increased yields and/or purity of isoprene.

Claims

1. Cells comprising (i) one or more non-modified nucleic acids encoding feedback-resistant mevalonate kinase polypeptides or (ii) one or more additional copies of an endogenous nucleic acid encoding a feedback-resistant mevalonate kinase polypeptide.

2. The cells of claim 1, wherein the feedback-resistant mevalonate kinase is archaeal mevalonate kinase.

3. The cells of claim 1, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

4. The cells of claim 1, wherein the heterologous nucleic acid encodes for an MVA pathway enzyme or a DXP pathway enzyme.

5. The cells of claim 4, wherein the MVA pathway enzyme is mevalonate kinase.

6. The cells of claim 2, wherein the feedback-resistant archaeal mevalonate kinase polypeptide is M. mazei mevalonate kinase.

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

8. The cells of claim 1, wherein the mevalonate kinase polypeptide is selected from the group consisting of Lactobacillus mevalonate kinase polypeptide, Lactobacillus sakei mevalonate kinase polypeptide, yeast mevalonate kinase polypeptide, Streptococcus mevalonate kinase polypeptide, Streptococcus pneumoniae mevalonate kinase polypeptide, and Streptomyces mevalonate kinase polypeptide, Streptomyces CL190 mevalonate kinase polypeptide.

9. The cells of claim 3, wherein the isoprene synthase polypeptide is from Pueraria or Populus or a hybrid, Populus alba×

Populus tremula.

10. The cells of claim 9, 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.

11. The cells of claim 1, wherein the cells are gram-positive bacterial cells, Streptomyces cells, gram-negative bacterial cells, Escherichia cells, Pantoea cells, fungal cells, filamentous fungal cells, Trichoderma cells, Aspergillus cells, or yeast cells.

12. The cells of claim 11, wherein the cells are selected from the group consisting of Bacillus subtilis, Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus, Escherichia coli, Pantoea citrea, Trichoderma reesei, Aspergillus oryzae and Aspergillus niger, Saccharomyces cerevisieae and Yarrowia lipolytica.

13. A composition for producing isoprene comprising cells of claim 1.

14. A method of producing isoprene, the method comprising

(a) culturing cells of claim 1 under suitable culture conditions for the production of isoprene, and

(b) producing isoprene.

15. The method of claim 14, wherein the cells further comprise one or more heterologous nucleic acids or one or more additional copies of an endogenous nucleic acid encoding an isoprene synthase polypeptide.

16. The method of claim 14, wherein the heterologous nucleic acid encodes for an MVA pathway enzyme or a DXP pathway enzyme.

17. The method of claim 16, wherein the MVA pathway enzyme is mevalonate kinase.

18. The method of claim 14, wherein the feedback-resistant mevalonate kinase is archaeal mevalonate kinase.

19. The method of claim 18, wherein the archaeal mevalonate kinase polypeptide is M. mazei mevalonate kinase.

20. The method of claim 14, wherein the cells in culture produce greater than about 400 nmole/g_wcm/hr of isoprene.

21. The method of claim 14, further comprising recovering the isoprene.

22. A method of increasing the rate or flux of production of 3,3-dimethylallyl diphosphate (DMAPP), isopentenyl diphosphate (IPP), or a product derived from 3,3-dimethylallyl diphosphate (DMAPP) or isopentenyl diphosphate (IPP) comprising:

(a) culturing cells of claim 1 wherein the cells are cultured under suitable culture conditions for increasing the rate or flux of production of 3,3-dimethylallyl diphosphate (DMAPP), isopentenyl diphosphate (IPP), or a product derived from 3,3-dimethylallyl diphosphate (DMAPP) or isopentenyl diphosphate (IPP), and

(b) producing 3,3-dimethylallyl diphosphate (DMAPP), isopentenyl diphosphate (IPP), or a product derived from 3,3-dimethylallyl diphosphate (DMAPP) or isopentenyl diphosphate (IPP).

23. The method of claim 22 wherein the product derived from 3,3-dimethylallyl diphosphate (DMAPP) or isopentenyl diphosphate (IPP) is isoprene.

24. The method of claim 22, wherein the feedback-resistant mevalonate kinase is archaeal mevalonate kinase.

25. The method of claim 24 wherein the archaeal mevalonate kinase polypeptide is M. mazei mevalonate kinase.

26. A system comprising a reactor chamber comprising cells of claim 1.

27. The system of claim 26 wherein the cells produce greater than about 400 nmole/g_wcm/hr isoprene.

28. A method of manufacturing a tire wherein the improvement comprises using the cells of claim 1 to produce isoprene for the manufacture of the tire.