Increased isoprene production using the archaeal lower mevalonate pathway

• US 9,464,301 B2
• Filed: 07/03/2014
• Issued: 10/11/2016
• Est. Priority Date: 09/15/2008
• Status: Expired due to Fees

• 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: Beck, Zachary Q., Calabria, Anthony R., Miller, Michael C., Nielsen, Alex T., Vaviline, Dmitrii V.
• Primary Examiner(s): Ariani, Kade
• Application Number:
  US14/323,596
• Publication Number:
  US 20150203873A1
• Time in Patent Office: 831 Days
• Field of Search: None
• US Class Current: 1/1
• 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 increasing the rate or flux of production of 3,3-dimethylallyl diphosphate (DMAPP), isopentenyl diphosphate (IPP), or a product derived from DMAPP and/or IPP comprising:

culturing recombinant cells comprising (i) one or more heterologous non-modified nucleic acids encoding feedback-resistant mevalonate kinase polypeptides, and (ii) one or more nucleic acids encoding a mevalonate (MVA) pathway polypeptide, wherein the cells are cultured under suitable culture conditions for increasing the rate or flux of production of DMAPP, IPP, or a product derived from DMAPP or IPP,

thereby increasing the rate or flux of production of DMAPP, IPP, or a product derived from DMAPP and/or IPP.

2. The method of claim 1, wherein the cells further comprise one or more nucleic acids encoding an isoprene synthase.

3. The method of claim 2, wherein the product derived from DMAPP and/or IPP is isoprene.

4. The method of claim 1, wherein the product derived from DMAPP and/or IPP is an isoprenoid.

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

6. The method of claim 5, wherein the archaeal mevalonate kinase polypeptide is an M. mazei mevalonate kinase.

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

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

9. The method of claim 3, wherein the cells produce greater than about 400 nmole/g_wcm/hr of isoprene.

10. 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.

11. The method of claim 10, wherein the cells are Bacillus cells, Saccharomyces cells, Streptomyces cells, Escherichia cells, Pantoea cells, Trichoderma cells, Yarrowia cells or Aspergillus cells.

12. 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 cerevisiae and Yarrowia lipolytica.

13. The method of claim 1, wherein the (ii) one or more nucleic acids encoding an 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) polypeptides, diphosphomevalonate decarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC) polypeptides, and isopentenyl phosphate kinase (IPK) polypeptides.

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

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

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

500 μ

M DMAPP.

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

20 μ

M geranyl diphosphate (GPP).

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

20 μ

M farnesyl diphosphate (FPP).

19. The method of claim 1, wherein the cells further comprise one or more nucleic acids encoding a non-mevalonate (DXP) pathway polypeptide.

20. The method of claim 1, wherein the cells are cultured in a culture medium comprising one or more carbon sources selected from the group consisting of a carbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon substrates, animal fat, animal oil, plant oil, fatty acid, phospholipid, glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbon source, polypeptide, and yeast extract.

21. The method of claim 1, wherein the cells are cultured in a culture medium comprising one or more carbon sources selected from the group consisting of oil and lipid.