Fermentive production of four carbon alcohols

1. A recombinant microbial host cell comprising heterologous DNA molecules encoding polypeptides that catalyze substrate to product conversions for each step below:

i) pyruvate to acetolactate;

ii) acetolactate to 2,3-dihydroxyisovalerate;

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate; and

iv) α-ketoisovalerate to isobutyraldehyde;

wherein said microbial host cell produces isobutanol; and wherein

a) the polypeptide that catalyzes a substrate to product conversion of pyruvate to acetolactate is acetolactate synthase having the EC number 2.2.1.6;

b) the polypeptide that catalyzes a substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate is acetohydroxy acid isomeroreductase having the EC number 1.1.1.86;

c) the polypeptide that catalyzes a substrate to product conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate is acetohydroxy acid dehydratase having the EC number 4.2.1.9;

d) the polypeptide that catalyzes a substrate to product conversion of α-ketoisovalerate to isobutyraldehyde is branched-chain α-keto acid decarboxylase having the EC number 4.1.1.72.

2. A host cell according to claim 1 wherein the cell is selected from the group consisting of: a bacterium, a cyanobacterium, a filamentous fungus and a yeast.

3. A host cell according to claim 2 wherein the cell is a member of a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

4. A host cell according to claim 3 wherein the cell is Escherichia coli.

5. A host cell according to claim 3 wherein the cell is Alcaligenes eutrophus.

6. A host cell according to claim 3 wherein the cell is Bacillus licheniformis.

7. A host cell according to claim 3 wherein the cell is Paenibacillus macerans.

8. A host cell according to claim 3 wherein the cell is Rhodococcus erythropolis.

9. A host cell according to claim 3 wherein the cell is Pseudomonas putida.

10. A host cell according to claim 3 wherein the cell is Bacillus subtilis.

11. A host cell according to claim 3 wherein the cell is Lactobacillus plantarum.

12. A host cell according to claim 3 wherein the cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium , and Enterococcus faecalis.

13. A host cell according to claim 3 wherein the cell is Saccharomyces cerevisiae.

14. A host cell according to claim 1 wherein the acetolactate synthase has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:178, and SEQ ID NO:180.

15. A host cell according to claim 1 wherein the acetohydroxy acid isomeroreductase has an amino acid sequence selected from the group consisting of SEQ ID NO:43, SEQ ID NO:181, SEQ ID NO:183, and SEQ ID NO:185.

16. A host cell according to claim 1 wherein the acetohydroxy acid dehydratase has an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:186, SEQ ID NO:188, and SEQ ID NO:190.

17. A host cell according to claim 1 wherein the host cell is a facultative anaerobe.

18. A method for the production of isobutanol comprising:

1) providing a recombinant microbial host cell of claim 1 ; and

2) contacting the host cell of (i) with a fermentable carbon substrate in a fermentation medium under conditions whereby isobutanol is produced.

19. A method according to claim 18 wherein the fermentable carbon substrate is selected from the group consisting of monosaccharides, oligosaccharides, and polysaccharides.

20. A method according to claim 18 wherein the carbon substrate is selected from the group consisting of glucose, sucrose, and fructose.

21. A method according to any of claim 18 wherein the conditions whereby isobutanol is produced are anaerobic.

22. A method according to any of claim 18 wherein the conditions whereby isobutanol is produced are microaerobic.

23. A method according to claim 18 wherein the host cell is contacted with the carbon substrate in a minimal medium.

24. A method according to claim 18 wherein the host cell is a member of a genus selected from the group consisting of Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula and Saccharomyces.

25. A method according to claim 24 wherein the host cell is Escherichia coli.

26. A method according to claim 24 wherein the host cell is Alcaligenes eutrophus.

27. A method according to claim 24 wherein the host cell is Bacillus licheniformis.

28. A method according to claim 24 wherein the host cell is Paenibacillus macerans.

29. A method according to claim 24 wherein the host cell is Rhodococcus erythropolis.

30. A method according to claim 24 wherein the host cell is Pseudomonas putida.

31. A method according to claim 24 wherein the host cell is Bacillus subtilis.

32. A method according to claim 24 wherein the host cell is Lactobacillus plantarum.

33. A method according to claim 24 wherein the host cell is selected from the group consisting of Enterococcus faecium, Enterococcus gallinarium , and Enterococcus faecalis.

34. A method according to claim 24 wherein the host cell is Saccharomyces cerevisiae.

35. An isobutanol containing fermentation medium produced by the method of claim 18 .

36. The host cell of claim 1 , wherein said host cell comprises at least one DNA molecule encoding a polypeptide that catalyzes conversion of isobutyraldehyde to isobutanol wherein said polypeptide is a branched-chain alcohol dehydrogenase having an EC number of 1.1.1.1, 1.1.1.2, or 1.1.1.265.

37. A host cell according to claim 36 , wherein said polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO:10, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, and SEQ ID NO:204.