Butanol tolerance in microorganisms

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.