Butanol tolerance in microorganisms

• US 9,273,330 B2
• Filed: 10/03/2013
• Issued: 03/01/2016
• Est. Priority Date: 10/03/2012
• Status: Active Grant

• Current Assignee: Gevo Incorporated
• Original Assignee: Butamax Advanced Biofuels LLC (International Flavors & Fragrances Incorporated)
• Inventors: Bramucci, Michael G., Nagarajan, Vasantha
• Primary Examiner(s): Raghu, Ganapathirama
• Application Number:
  US14/045,506
• Publication Number:
  US 20140096439A1
• Time in Patent Office: 880 Days
• Field of Search: 444/51, 435/157, 435/160, 435/254.2, 435/254.21, 435/254.22, 435/254.23
• US Class Current: 1/1
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12P 7/04 acyclic
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12Y 401/01001 Pyruvate decarboxylase (4.1.1.1)
• CPC Class Codes: C12Y 406/01001 Aodenylate cyclase (4.6.1.1)
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Provided herein are recombinant yeast host cells and methods for their use for production of fermentation products from a pyruvate utilizing pathway. Yeast host cells provided herein comprise reduced pyruvate decarboxylase activity and modified adenylate cyclase activity. In embodiments, yeast host cells provided herein comprise resistance to butanol and increased biomass production.

Claims

1. A recombinant yeast microorganism comprising a pyruvate utilizing biosynthetic pathway, wherein the pyruvate utilizing biosynthetic pathway is an isobutanol biosynthetic pathway comprising heterologous DNA molecules encoding polypeptides that catalyze the following substrate to product conversions:

a) pyruvate to acetolactate;

b) acetolactate to 2,3-dihydroxyisovalerate;

c) 2,3-dihydroxyisovalerate to α

-ketoisovalerate;

d) α

-ketoisovalerate to isobutyraldehyde; and

e) isobutyraldehyde to isobutanol; and

wherein the recombinant yeast microorganism further comprises a first genetic modification, wherein the first genetic modification is a deletion or disruption in at least one endogenous gene encoding a pyruvate decarboxylase enzyme, wherein the deletion or disruption eliminates pyruvate decarboxylase activity, and

wherein the recombinant yeast microorganism further comprises a second genetic modification, wherein the second genetic modification is a substitution mutation in an adenylate cyclase gene, wherein the substitution mutation is at a residue equivalent to A1814 or H1873 of SEQ ID NO;

1 and wherein the adenylate cyclase gene encodes a polypeptide having at least 90% sequence identity to SEQ ID NO;

1.

2. The microorganism of claim 1, wherein the pyruvate decarboxylase gene is PDC1, PDC5, PDC6, or combinations thereof.

3. The microorganism of claim 1, further comprising a deletion, disruption, or mutation in a gene that regulates adenylate cyclase activity.

4. The microorganism of claim 3, wherein the gene is GPR1, GPA2, RAS1, RAS2, or combinations thereof.

5. The microorganism of claim 1 comprising:

a. at least one gene encoding an adenylate cyclase comprising the amino acid sequence of SEQ ID NO;

2;

b. at least one gene encoding an adenylate cyclase comprising the amino acid sequence of SEQ ID NO;

3;

or

c. both a) and b).

6. The microorganism of claim 1 which further comprises a mutation in a gene selected from the group consisting of FLO9, NUM1, PAU10, YGR109W-B, HSP32, ATG13, and combinations thereof.

7. The microorganism of claim 1, wherein the microorganism is a member of a genus selected from the group consisting of Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Kluyveromyces, Yarrowia, Issatchenkia, and Pichia.

8. The microorganism of claim 1, wherein the microorganism has an increased biomass yield as compared to a corresponding microorganism that does not have the substitution mutation in the adenylate cyclase gene, wherein the substitution mutation is at a residue equivalent to A1814 or H1873 of SEQ ID NO:

1 and wherein the adenylate cyclase gene encodes a polypeptide having at least 90% sequence identity to SEQ ID NO;

1.

9. The microorganism of claim 1, wherein carbon flow through the pyruvate utilizing pathway is not reduced compared to a corresponding microorganism that does not have the substitution mutation in the adenylate cyclase gene, wherein the substitution mutation is at a residue equivalent to A1814 or H1873 of SEQ ID NO:

1 and wherein the adenylate cyclase gene encodes a polypeptide having at least 90% sequence identity to SEQ ID NO;

1.

10. A method of producing a fermentation product from a pyruvate utilizing biosynthetic pathway, wherein the pyruvate utilizing biosynthetic pathway is an isobutanol biosynthetic pathway, comprising:

a. providing the microorganism according to claim 1; and

b. growing the microorganism under conditions whereby isobutanol is produced from pyruvate.

11. The method of claim 10, further comprising (c) recovering the isobutanol.

12. The method of claim 11 further comprising (d) removing solids from the fermentation medium.