Reduction of carbon dioxide emission during isoprene production by fermentation

• US 8,470,581 B2
• Filed: 09/15/2009
• Issued: 06/25/2013
• Est. Priority Date: 09/15/2008
• Status: Expired due to Fees

• Current Assignee: Goodyear Tire & Rubber Company
• Original Assignee: Goodyear Tire & Rubber Company, Danisco USA Incorporated (International Flavors & Fragrances Incorporated)
• Inventors: Chotani, Gopal K., Sanford, Karl J., Nielsen, Alex
• Primary Examiner(s): DESAI, ANAND U
• Application Number:
  US12/560,317
• Publication Number:
  US 20100167370A1
• Time in Patent Office: 1,379 Days
• Field of Search: None
• US Class Current: 435/252.3
• CPC Class Codes: C12N 1/20 Bacteria; Culture media therefor
• CPC Class Codes: C12P 5/007 containing one or more isoprene units, i.e. terpenes carotenes C12P23/00
• CPC Class Codes: Y02E 50/30 Fuel from waste, e.g. synthetic alcohol or diesel

Abstract

The present invention provides methods for increasing the amount of isoprene produced by cultured cells with only a minimal increase in carbon dioxide emitted, thereby resulting in process having a greater yield of isoprene relative to carbon dioxide. In addition, the present invention provides compositions that include the cultured cells or isoprene produced there from.

Claims

1. A cell culture comprising cells capable of producing isoprene, wherein said cell culture comprises:

(a) recombinant cells comprising a heterologous nucleic acid that encodes an isoprene synthase polypeptide in operable combination with a promoter;

(b) culture medium comprising one or more uncommon carbon sources; and

(c) culture conditions comprising an inlet air flow rate of less than or equal to 0.4 volume(air)/volume (culture medium)/minute (vvm);

wherein said cell culture produces isoprene and carbon dioxide and the ratio of carbon dioxide emitted per isoprene produced in off gas from the cell culture is less than 500.

2. The cell culture of claim 1, wherein the uncommon carbon source is selected from the group consisting of glycerol, cell mass, protein, alcohol, and plant-derived oil.

3. The cell culture of claim 1, wherein the cells further comprise one or more heterologous nucleic acids that encode for an MVA pathway enzyme or a DXP pathway enzyme.

4. The cells of claim 3, wherein the MVA pathway enzyme is mevalonate kinase.

5. The cells of claim 4, wherein the mevalonate kinase is selected from the group consisting of feedback-resistant mevalonate kinase, archaeal mevalonate kinase, M. mazei mevalonate kinase, Lactobacillus mevalonate kinase polypeptide, Lactobacillus sakei mevalonate kinase polypeptide, yeast mevalonate kinase polypeptide, Saccharomyces cerevisiae mevalonate kinase polypeptide, Streptococcus mevalonate kinase polypeptide, Streptococcus pneumoniae mevalonate kinase polypeptide, and Streptomyces mevalonate kinase polypeptide, Streptomyces CL190 mevalonate kinase polypeptide.

6. The cell culture of claim 1, wherein at least a portion of the cells maintain the isoprene synthase nucleic acid for at least or about 5, 10, 20, 40, 50, 60, 65, or more cell divisions in a continuous culture.

7. The cell culture of claim 1, wherein the isoprene synthase polypeptide is from Pueraria or Populus or a hybrid, Populus alba×

Populus tremula.

8. The cell culture of claim 7, 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.

9. The cell culture 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.

10. The cell culture of claim 9, 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, Yarrowia lipolytica and Saccharomyces cerevisieae.

11. A composition for producing isoprene comprising the cell culture of claim 1.

12. A method of producing isoprene, the method comprising:

(a) culturing cells of claim 1 in culture medium under conditions suitable for the production of isoprene, wherein the culture medium comprises one or more uncommon carbon sources and the cells comprise a heterologous nucleic acid that encodes an isoprene synthase polypeptide in operable combination with a promoter; and

(b) producing isoprene, wherein the ratio of carbon dioxide emitted per isoprene produced in off gas from the cultured cells is less than 500.

13. The method of claim 12 wherein the uncommon carbon source is selected from the group consisting of glycerol, cell mass, protein, alcohol, and plant-derived oil.

14. The method of claim 13, wherein the alcohol comprises methanol.

15. The method of claim 12, wherein the isoprene produced as a percentage of off gas from the cultured cells is greater than 0.03%.

16. The method of claim 12, further comprising recovering the isoprene.