Fermentive production of four carbon alcohols

1. A recombinant microbial host cell comprising genes encoding an engineered isobutanol biosynthetic pathway comprising the following substrate to product conversions:

i) pyruvate to acetolactate, (pathway step a)

ii) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c)

iv) α-ketoisovalerate to valine, (pathway step h)

v) valine to isobutylamine, (pathway step i)

vi) isobutylamine to isobutyraldehyde, (pathway step j) and

vii) isobutyraldehyde to isobutanol; (pathway step e)

wherein the substrate to product conversions of i)-vi) are encoded by genes that are heterologous to said recombinant microbial host cell and wherein said microbial host cell produces isobutanol.

2. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of pyruvate to acetolactate of step (a) is performed by a recombinantly expressed acetolactate synthase.

3. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of acetolactate to 2,3-dihydroxyisovalerate of step (b) is performed by a recombinantly expressed acetohydroxy acid isomeroreductase.

4. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of 2,3-dihydroxyisovalerate to α-ketoisovalerate of step (c) is performed by a recombinantly expressed acetohydroxy acid dehydratase.

5. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of isobutyraldehyde to isobutanol of pathway step (e) is encoded by a gene that is heterologous to said recombinant microbial host cell.

6. The recombinant microbial host cell according to claim 5 wherein the substrate to product conversion of isobutyraldehyde to isobutanol of step (e) is performed by a recombinantly expressed branched-chain alcohol dehydrogenase.

7. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of α-ketoisovalerate to valine of step (h) is performed by a recombinantly expressed valine transaminase.

8. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of α-ketoisovalerate to valine of step (h) is performed by a recombinantly expressed valine dehydrogenase.

9. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of valine to isobutylamine of step (i) is performed by a recombinantly expressed valine decarboxylase.

10. The recombinant microbial host cell according to claim 1 wherein the substrate to product conversion of isobutylamine to isobutyraldehyde of step (j) is performed by a recombinantly expressed omega transaminase.

11. The recombinant microbial 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.

12. A method for the production of isobutanol comprising:

1) providing a recombinant microbial host cell comprising genes encoding an engineered isobutanol biosynthetic pathway comprising the following substrate to product conversions:

i) pyruvate to acetolactate, (pathway step a)

i) acetolactate to 2,3-dihydroxyisovalerate, (pathway step b)

iii) 2,3-dihydroxyisovalerate to α-ketoisovalerate, (pathway step c)

iv) α-ketoisovalerate to valine, (pathway step h)

v) valine to isobutylamine, (pathway step i)

vi) isobutylamine to isobutyraldehyde, (pathway step j) and

isobutyraldehyde to isobutanol; (pathway step e)

wherein the substrate to produce conversions of i)-vi) are encoded by genes that are heterologous to said recombinant microbial host cell; and

2) contacting the recombinant microbial host cell of 1) with a fermentable carbon in a fermentation medium substrate under conditions whereby isobutanol is produced.

13. An isobutanol containing fermentation medium produced by the method of claim 12 .

14. The recombinant microbial host cell of claim 1 wherein the host cell also produces fusel oils.

15. The recombinant microbial host cell of claim 1 further comprising an inactivated gene thereby reducing yield loss from competing pathways for carbon flow.

16. The recombinant microbial host cell of claim 2 wherein the recombinantly expressed acetolactate synthase is from Bacillus subtilis, Klebsiella pneumoniae , or Lactococcus lactis.

17. The recombinant microbial host cell of claim 3 wherein the recombinantly expressed acetohydroxy acid isomeroreductase is from Escherichia coil, Saccharomyces cerevisiae, Methanococcus maripaludis , or Bacillus subtilis.

18. The recombinant microbial host cell of claim 4 wherein the recombinantly expressed acetohydroxy acid dehydratase is from Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae , or Methanococcus maripaludis.

19. The recombinant microbial host cell of claim 6 wherein the recombinantly expressed branched-chain alcohol dehydrogenase is from Saccharomyces cerevisiae, Escherichia coli , or Clostridium acetobutylicum.

20. The recombinant microbial host cell of claim 7 wherein the recombinantly expressed valine transaminase is from Escherichia coli, Bacillus lichenformis, Saccharomyces cerevisiae , or Methanobacterium thermoautotrophicum.

21. The recombinant microbial host cell of claim 8 wherein the recombinantly expressed valine dehydrogenase is from Streptomyces coelicolor or Bacillus subtilis.

22. The recombinant microbial host cell of claim 9 wherein the recombinantly expressed valine decarboxylase is from Streptomyces viridifaciens.

23. The recombinant microbial host cell of claim 10 wherein the recombinantly expressed omega transaminase is from Alcaligenes denitrificans, Ralstonia eutropha, Shewanella oneidensis , or Pseudomonas putida.