Compositions and methods for producing isoprene free of C5 hydrocarbons under decoupling conditions and/or safe operating ranges

Patent metadata

Field	Value
Publication number	US-9777294-B2
Application number	US-201514973485-A
Country	US
Kind code	B2
Filing date	Dec 17, 2015
Priority date	Jul 2, 2008
Publication date	Oct 3, 2017
Grant date	Oct 3, 2017

Abstract

The invention features methods for producing isoprene from cultured cells wherein the cells in the stationary phase. The invention also provides compositions that include these cultured cells and/or increased amount of isoprene. The invention also provides for systems that include a non-flammable concentration of isoprene in the gas phase. Additionally, the invention provides isoprene compositions, such as compositions with increased amount of isoprene or increased purity.

First claim

What is claimed is:

1. A method of producing isoprene, the method comprising:
(a) culturing recombinant cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under limited glucose conditions for the production of isoprene, wherein the amount of isoprene produced during the stationary phase is greater than 2-fold more than the amount of isoprene produced during the growth phase;
(b) producing isoprene; and
(c) recovering the isoprene made in step (b).

2. The method of claim 1 , wherein the isoprene is recovered from an off-gas portion of the cell culture.

3. The method of claim 1 , wherein the isoprene synthase polypeptide is a plant isoprene synthase polypeptide.

4. The method of claim 1 , wherein the cells are bacterial cells.

5. The method of claim 4 , wherein the bacterial cells are selected from the group consisting of Escherichia coli, Pantoae citrea, Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus clausii, Bacillus halodurans, Bacillus megaterium, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus thuringiensis, Streptomyces lividans, Streptomyces coelicolor, Streptomyces griseus, Pseudomonas sp., and Pseudomonas alcaligenes cells.

6. The method of claim 1 , wherein the cells are fungal cells.

7. The method of claim 6 , wherein the fungal cells are selected from the group consisting of Aspergillus sp., yeast, Trichoderma sp., Yarrowia sp., Saccharomyces sp., Schizosaccharomyces sp., Pichia sp., Candida sp., Aspergillus oryzae, Aspergillus niger, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Trichoderma reesei, H. insolens, H. lanuginose, H. grisea, Candida lucknowense, Aspergillus sojae, Aspergillus japonicus, Aspergillus nidulans, Aspergillus aculeatus, Aspergillus awamori, Fusarium roseum, Fusarium graminum, Fusarium cerealis, Fusarium oxysporuim, F. venenatum, Neurospora crassa, Mucor miehei, Trichoderma viride, Fusarium oxysporum , and Fusarium solani cells.

8. The method of claim 1 , wherein the cells are algal cells.

9. The method of claim 8 , wherein the algal cells are selected from the group consisting of green algae, red algae, glaucophytes, chlorarachniophytes, euglenids, chromista, and dinoflagellates.

10. The method of claim 1 , wherein the cells further comprise a nucleic acid encoding an isopentyl-diphosphate delta-isomerase (IDI) polypeptide.

11. The method of claim 1 , wherein the cells further comprise a heterologous nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase (DXS) polypeptide.

12. The method of claim 1 , wherein the cells further comprise one or more nucleic acids encoding one or more mevalonate (MVA) pathway polypeptides.

13. The method of claim 12 , wherein the cells further comprise one or more heterologous nucleic acids encoding one or more MVA pathway polypeptides.

14. The method of claim 12 , wherein the cells further comprise one or more nucleic acids encoding the polypeptides of the entire MVA pathway.

15. The method of claim 1 , wherein the cells further comprise:
(a) a nucleic acid encoding an isopentyl-diphosphate delta-isomerase (IDI) polypeptide; and (b) (i) a nucleic acid encoding a 1-deoxyxylulose-5-phosphate synthase (DXS) polypeptide and/or (ii) one or more nucleic acids encoding one or more mevalonate (MVA) pathway polypeptides.

16. The method of claim 1 , wherein the cells are cultured in a medium comprising a carbon source, and wherein the cells convert at least 0.002% of the carbon source into isoprene.

17. The method of claim 1 , wherein the acetate kinase (ack) and phosphate acetyl transferase (pta) genes have been deleted from the cells.

18. The method of claim 1 , wherein greater than 90% of the total amount of isoprene that is produced is produced while the cells divide slowly or not at all such that the optical density at 550 nm of the cells increases by less than 50% during the stationary phase.

19. A method of producing a vessel comprising isoprene, the method comprising:
(a) culturing recombinant cells comprising a heterologous nucleic acid encoding an isoprene synthase polypeptide under limited glucose conditions for the production of isoprene, wherein the amount of isoprene produced during the stationary phase is greater than 2-fold more than the amount of isoprene produced during the growth phase; and
(b) producing the isoprene.

Assignees

• Danisco Us Inc
• Goodyear Tire & Rubber

Inventors

• Calabria Anthony R
• Cervin Marguerite A
• Chotani Gopal K
• La Duca Richard
• Mcauliffe Joseph C
• Miller Michael C
• Sabo Timothy A
• Sanford Karl J
• Spring Erin L
• Whited Gregory M

Classifications

• C07C11/18

  Isoprene · CPC title

• C12P5/007 Primary

  containing one or more isoprene units, i.e. terpenes (carotenes C12P23/00) · CPC title

• Y02E50/343

  Cross-Sectional Technologies · mapped topic

• C12N15/52

  Genes encoding for enzymes or proenzymes · CPC title

• C12N9/88

  Lyases (4.) · CPC title