Host cells and methods for production of isobutanol

• US 9,422,582 B2
• Filed: 03/15/2013
• Issued: 08/23/2016
• Est. Priority Date: 03/24/2011
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax(TM) Advanced Biofuels LLC (BP PLC)
• Inventors: Anthony, Larry Cameron, He, Hongxian, Huang, Lixuan Lisa, Okeefe, Daniel P., Kruckeberg, Arthur Leo, Li, Yougen, Maggio-Hall, Lori Ann, McElvain, Jessica, Nelson, Mark J., Patnaik, Ranjan, Rothman, Steven Cary
• Primary Examiner(s): HOLLAND, PAUL J
• Application Number:
  US13/840,676
• Publication Number:
  US 20140030783A1
• Time in Patent Office: 1,257 Days
• Field of Search: 435/160
• US Class Current: 1/1
• CPC Class Codes: C12N 1/38 Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound C12N1/34 takes precedence
• CPC Class Codes: C12N 15/81 for yeasts
• CPC Class Codes: C12N 15/815 for yeasts other than Saccharomyces
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/42 Hydroxy-carboxylic acids
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel
• CPC Class Codes: Y02P 20/52 using catalysts, e.g. selective catalysts

Abstract

Provided herein are recombinant yeast host cells and methods for their use for production of isobutanol. Yeast host cells provided comprise an isobutanol biosynthetic pathway and at least one of reduced or eliminated aldehyde dehydrogenase activity, reduced or eliminated acetolactate reductase activity; or a heterologous polynucleotide encoding a polypeptide having ketol-acid reductoisomerase activity.

Claims

1. A method for producing isobutanol comprising:

a. providing a recombinant yeast cell comprising;

(i) an engineered isobutanol production pathway comprising a heterologous polypeptide having ketol-acid reductoisomerase (KARI) activity, wherein said polypeptide having KARI activity has at least 99% identity to the amino acid sequence set forth in SEQ ID NO;

419;

(ii) a modification that eliminates expression or activity of a pyruvate decarboxylase polypeptide, wherein said modification is a deletion of an endogenous gene encoding said pyruvate decarboxylase polypeptide;

(iii) a modification that eliminates expression or activity of an NAD-dependent glycerol-3-phosphate dehydrogenase polypeptide, wherein said modification is a deletion of an endogenous gene encoding said NAD-dependent glycerol-3-phosphate dehydrogenase polypeptide; and

(iv) a modification that eliminates expression or activity of a polypeptide affecting Fe—

S cluster biosynthesis in the yeast, wherein said modification is a deletion of an endogenous gene encoding said polypeptide affecting Fe—

S cluster biosynthesis in yeast; and

b. contacting said recombinant yeast cell of a) with a carbon substrate under conditions whereby isobutanol is produced;

wherein at least a portion of said contacting occurs under anaerobic conditions.

2. The method of claim 1, wherein said recombinant yeast cell comprises a cytosol-localized acetolactate synthase.

3. The method of claim 1, wherein the isobutanol is removed as it is produced.

4. The method of claim 3, wherein said recombinant yeast cell is contacted with an organic extractant.

5. The method of claim 4, wherein said organic extractant comprises C₁₂ to C₂₂ fatty acids.

6. The method of claim 1, wherein the molar ratio of isobutanol to glycerol produced is greater than 1.

7. The method of claim 1, wherein said recombinant yeast cell further comprises a modification that eliminates expression or activity of an acetolactate reductase polypeptide in the yeast, wherein said modification is a deletion of an endogenous gene encoding said acetolactate reductase polypeptide.

8. The method of claim 1, wherein said recombinant yeast cell further comprises a modification that eliminates expression or activity of an aldehyde dehydrogenase polypeptide in the yeast, wherein said modification is a deletion of an endogenous gene encoding said aldehyde dehydrogenase polypeptide.

9. A method for producing isobutanol comprising:

a. providing a recombinant yeast cell comprising;

(i) an engineered isobutanol production pathway comprising a heterologous polypeptide having ketol-acid reductoisomerase (KARI) activity, wherein said polypeptide comprises the amino acid sequence set forth in SEQ ID NO 419;

(ii) a modification that eliminates expression or activity of a pyruvate decarboxylase polypeptide, wherein said modification is a deletion of an endogenous gene encoding said pyruvate decarboxylase polypeptide;

(iii) a modification that eliminates expression or activity of an NAD-dependent glycerol-3-phosphate dehydrogenase polypeptide, wherein said modification is a deletion of an endogenous gene encoding said NAD-dependent glycerol-3-phosphate dehydrogenase polypeptide; and

(iv) a modification that eliminates expression or activity of a polypeptide affecting Fe—

S cluster biosynthesis in the yeast, wherein said modification is a deletion of an endogenous gene encoding said polypeptide affecting Fe—

S cluster biosynthesis in yeast; and

b. contacting said recombinant yeast cell of a) with a carbon substrate under conditions whereby isobutanol is produced;

wherein at least a portion of the said contacting occurs under anaerobic conditions.

10. The method of claim 9, wherein said recombinant yeast cell further comprises a modification that eliminates expression or activity of an acetolactate reductase polypeptide in the yeast, wherein said modification is a deletion of an endogenous gene encoding said acetolactate reductase polypeptide.

11. The method of claim 9, wherein said recombinant yeast cell further comprises a modification that eliminates expression or activity of an aldehyde dehydrogenase polypeptide in the yeast, wherein said modification is a deletion of an endogenous gene encoding said aldehyde dehydrogenase polypeptide.

12. The method of claim 9, wherein said recombinant yeast cell comprises a cytosol-localized acetolactate synthase.

13. The method of claim 9, wherein the isobutanol is removed as it is produced.

14. The method of claim 13, wherein said recombinant yeast cell is contacted with an organic extractant.

15. The method of claim 14, wherein said organic extractant comprises C₁₂ to C₂₂ fatty acids.

16. The method of claim 9, wherein the molar ratio of isobutanol to glycerol produced is greater than 1.