CYTOSOLIC ISOBUTANOL PATHWAY LOCALIZATION FOR THE PRODUCTION OF ISOBUTANOL

US 20110076733A1
Filed: 08/12/2010
Published: 03/31/2011
Est. Priority Date: 08/12/2009
Status: Active Grant

First Claim

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1. A recombinant microorganism comprising an isobutanol-producing metabolic pathway, wherein said isobutanol-producing comprises five isobutanol pathway enzymes localized in the cytosol, and wherein at least one of the five isobutanol pathway enzymes is a heterologous cytosolically active dihydroxyacid dehydratase (DHAD) enzyme.

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Abstract

The present invention provides recombinant microorganisms comprising isobutanol producing metabolic pathway with at least one isobutanol pathway enzyme localized in the cytosol, wherein said recombinant microorganism is selected to produce isobutanol from a carbon source. Methods of using said recombinant microorganisms to produce isobutanol are also provided. In various aspects of the invention, the recombinant microorganisms may comprise a cytosolically active isobutanol pathway enzymes. In some embodiments, the invention provides mutated, modified, and/or chimeric isobutanol pathway enzymes with cytosolic activity. In various embodiments described herein, the recombinant microorganisms may be microorganisms of the Saccharomyces clade, Crabtree-negative yeast microorganisms, Crabtree-positive yeast microorganisms, post-WGD (whole genome duplication) yeast microorganisms, pre-WGD (whole genome duplication) yeast microorganisms, and non-fermenting yeast microorganisms.

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28 Claims

1. A recombinant microorganism comprising an isobutanol-producing metabolic pathway, wherein said isobutanol-producing comprises five isobutanol pathway enzymes localized in the cytosol, and wherein at least one of the five isobutanol pathway enzymes is a heterologous cytosolically active dihydroxyacid dehydratase (DHAD) enzyme.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28)
- - 2. The recombinant microorganism of claim 1, wherein said DHAD enzyme comprises the amino acid sequence P(I/L)XXXGX(I/L)XIL (SEQ ID NO:
    - 27), wherein X is any natural or non-natural amino acid.
  - 3. The recombinant microorganism of claim 1, wherein said DHAD enzyme is derived from a bacterial organism.
  - 4. The recombinant microorganism of claim 3, wherein said bacterial organism is Lactococcus lactis.
  - 5. The recombinant microorganism of claim 4, wherein said DHAD enzyme from L. lactis comprises the amino acid sequence of SEQ ID NO:
    - 18.
  - 6. The recombinant microorganism of claim 1, wherein said DHAD enzyme is derived from a eukaryotic organism.
  - 7. The recombinant microorganism of claim 6, wherein said eukaryotic organism is Neurospora crassa.
  - 8. The recombinant microorganism of claim 7, wherein said DHAD enzyme from N. crassa comprises the amino acid sequence of SEQ ID NO:
    - 165.
  - 9. The recombinant microorganism of claim 1, wherein said recombinant microorganism exhibits at least about 50 percent greater DHAD activity in the cytosol as compared to a parental microorganism that lacks the heterologous cytosolically active DHAD enzyme.
  - 10. The recombinant microorganism of claim 1, wherein said DHAD exhibits a specific cytosolic activity which is greater than the specific mitochondrial activity.
  - 11. The recombinant microorganism of claim 1, wherein the five isobutanol pathway enzymes are selected from acetolactate synthase (ALS), ketol-acid reductoisomerase (KARI), dihydroxyacid dehydratase (DHAD), 2-keto-acid decarboxylase (KIVD), and alcohol dehydrogenase (ADH).
  - 12. The recombinant microorganism of claim 11, wherein said isobutanol pathway enzyme is acetolactate synthase (ALS).
  - 13. The recombinant microorganism of claim 12, wherein said ALS is derived from a prokaryotic organism.
  - 14. The recombinant microorganism of claim 13, wherein said prokaryotic organism is selected from Bacillus subtilis and L. lactis.
  - 15. The recombinant microorganism of claim 12, wherein said ALS is derived from a eukaryotic organism.
  - 16. The recombinant microorganism of claim 15, wherein said eukaryotic organism is selected from M. grisea, P. nodorum, T. stipitatus, and T. atroviride.
  - 17. The recombinant microorganism of claim 11, wherein said isobutanol pathway enzyme is ketol-acid reductoisomerase (KARI).
  - 18. The recombinant microorganism of claim 17, wherein said KARI is derived from a prokaryotic organism.
  - 19. The recombinant microorganism of claim 18, wherein said prokaryotic organism is selected from Escherichia coli, B. subtilis, and L. lactis.
  - 20. The recombinant microorganism of claim 17, wherein said KARI is derived from a eukaryotic organism.
  - 21. The recombinant microorganism of claim 20, wherein said eukaryotic organism is selected from Piromyces sp. E2, Saccharomyces cerevisiae, and Arabidopsis.
  - 22. The recombinant microorganism of claim 17, wherein said KARI comprises an amino acid sequence selected from the group consisting of SEQ ID NOs:
    - 1-8.
  - 23. The recombinant microorganism of claim 17, wherein said KARI is an NADH-dependent KARI.
  - 24. The recombinant microorganism of claim 23, wherein said KARI comprises the amino acid sequence of SEQ ID NO:
    - 170.
  - 25. The recombinant microorganism of claim 1, wherein said recombinant microorganism is a yeast microorganism.
  - 26. The recombinant microorganism of claim 25, wherein said yeast microorganism is selected from a Saccharomyces sensu stricto microorganism, a Crabtree-negative microorganism, Crabtree-positive microorganism, a pre-WGD microorganism, a post-WGD microorganism, a non-fermenting microorganism, or an organism of the Saccharomyces clade.
  - 27. The recombinant microorganism of claim 25, wherein said yeast microorganism is selected from S. cerevisiae, Kluyveromyces marxianus, Kluyveromyces lactis, or Issatchenkia orientalis.
  - 28. A method of producing isobutanol, comprising:
    - (a) providing a recombinant microorganism according to claim 1; and
      
      (b) cultivating said recombinant microorganism in a culture medium containing a feedstock providing the carbon source, until a recoverable quantity of the isobutanol is produced.

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Current Assignee
Gevo Incorporated
Original Assignee
Gevo Incorporated
Inventors
Aristidou, Aristos, Hawkins, Andrew, Feldman, Reid M. Renny, Berry, Ruth, Buelter, Thomas, Kalra, Ishmeet, Porter-Scheinman, Stephanie, Meinhold, Peter, Lies, Doug, Urano, Jun, Peters, Matthew, Dundon, Catherine Asleson

Granted Patent

US 8,232,089 B2
Time in Patent Office

Days
Field of Search
US Class Current

435/160
CPC Class Codes

C12N 15/81   for yeasts

C12N 9/0006   acting on CH-OH groups as d...

C12N 9/1022   transferring aldehyde or ke...

C12N 9/88   Lyases (4.)

C12P 7/16   Butanols

Y02E 50/10   Biofuels, e.g. bio-diesel

CYTOSOLIC ISOBUTANOL PATHWAY LOCALIZATION FOR THE PRODUCTION OF ISOBUTANOL

First Claim

13 Assignments

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Abstract

67 Citations

28 Claims

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CYTOSOLIC ISOBUTANOL PATHWAY LOCALIZATION FOR THE PRODUCTION OF ISOBUTANOL

First Claim

13 Assignments

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0 Petitions

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Accused Products

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Abstract

67 Citations

28 Claims

Specification

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Solutions

Use Cases

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