Increased isoprene production using the archaeal lower mevalonate pathway

• US 8,815,548 B2
• Filed: 12/28/2012
• Issued: 08/26/2014
• Est. Priority Date: 09/15/2008
• Status: Active Grant

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Beck, Zachary Q., Calabria, Anthony R., Miller, Michael C., Nielsen, Alex T., Vaviline, Dmitrii V.
• Primary Examiner(s): Ariani, Kade
• Application Number:
  US13/730,661
• Publication Number:
  US 20130280769A1
• Time in Patent Office: 606 Days
• Field of Search: None
• 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. A method of producing isoprene, the method comprising

(a) culturing recombinant cells comprising (i) one or more heterologous nucleic acids encoding feedback-resistant mevalonate kinase polypeptides, (ii) one or more heterologous nucleic acids encoding an isoprene synthase polypeptide or one or more additional copies of an endogenous nucleic acid encoding an isoprene synthase polypeptide, and (iii) one or more nucleic acids encoding a mevalonate (MVA) pathway polypeptide under suitable culture conditions for the production of isoprene, and

(b) producing isoprene.

2. The method of claim 1, wherein the cells further comprise (iv) one or more nucleic acids encoding a DXP pathway enzyme.

3. The method of claim 1, wherein the feedback-resistant mevalonate kinase is archaeal mevalonate kinase.

4. The method of claim 3, wherein the archaeal mevalonate kinase polypeptide is M. mazei mevalonate kinase.

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

6. The method of claim 1, further comprising recovering the isoprene.

7. The method of claim 1, wherein the (iii) one or more nucleic acids encoding a mevalonate (MVA) pathway polypeptide is heterologous.

8. The method of claim 1, wherein the (iii) one or more nucleic acids encoding a mevalonate (MVA) pathway polypeptide is endogenous.

9. The method of claim 1, wherein the isoprene synthase polypeptide is from Pueraria or Populus or a hybrid, Populus albax Populus tremula, or mutant thereof.

10. The method 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, or mutant thereof.

11. The method of claim 1, wherein the cells are gram-positive bacterial cells, gram-negative bacterial cells, fungal cells, filamentous fungal cells, or yeast cells.

12. The method of claim 11, wherein the cells are Streptomyces cells, Escherichia cells, Pantoea cells, Trichoderma cells, or Aspergillus cells.

13. The method 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, Aspergillus niger, Saccharomyces cerevisieae and Yarrowia lipolytica.

14. The method of claim 1, wherein the (iii) one or more nucleic acids encoding a mevalonate (MVA) pathway polypeptide is selected from the group consisting of acetyl-CoA acetyltransferase (AA-CoA thiolase) polypeptide, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase) polypeptide, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) polypeptide, mevalonate kinase (MVK) polypeptide, phosphomevalonate kinase (PMK) polypeptide, diphosphomevalonate decarboxylase (MVD) polypeptide, phosphomevalonate decarboxylase (PMDC) polypeptide, isopentenyl phosphate kinase (IPK) polypeptide.

15. The method of claim 1, wherein the (iii) one or more nucleic acids encoding a mevalonate (MVA) pathway polypeptide encodes for upper MVA pathway polypeptide selected from the group consisting of AA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase polypeptide.

16. The method of claim 1, wherein the (iii) one or more nucleic acids encoding a mevalonate (MVA) pathway polypeptide encodes for lower MVA pathway polypeptide selected from the group consisting of MVK, PMK, MVD, and IDI (Isopentenyl-diphosphate delta isomerase) polypeptide.

17. The method of claim 1, wherein the feedback-resistant mevalonate kinase polypeptide has a K_i≧

500 μ

M DMAPP.

18. The method of claim 1, wherein the feedback-resistant mevalonate kinase polypeptide has a K_i≧

20 μ

M GPP.

19. The method of claim 1, wherein the feedback-resistant mevalonate kinase polypeptide has a K_i≧

20 μ

M FPP.

20. The method of claim 1, wherein the (i) one or more heterologous nucleic acids encoding feedback-resistant mevalonate kinase polypeptides are non-modified.