Fermentive production of four carbon alcohols

1. A recombinant microbial host cell comprising an engineered isobutanol biosynthetic pathway, wherein said isobutanol biosynthetic pathway comprises the following substrate to product conversions:

(a) pyruvate to acetolactate;

(b) the acetolactate from (a) to 2,3-dihydroxy-isovalerate;

(c) the 2,3-dihydroxy-isovalerate from (b) to α-ketoisovalerate;

(d) the α-ketoisovalerate from (c) to isobutyraldehyde; and

(e) the isobutyraldehyde from (d) to isobutanol;

wherein

i) the substrate to product conversion of step (a) is performed by a recombinantly expressed acetolactate synthase enzyme;

ii) the substrate to product conversion of step (b) is performed by a recombinantly expressed acetohydroxy acid isomeroreductase enzyme;

iii) the substrate to product conversion of step (c) is performed by a recombinantly expressed acetohydroxy acid dehydratase enzyme;

iv) the substrate to product conversion of step (d) is performed by a recombinantly expressed decarboxylase enzyme; and

v) the substrate to product conversion of step (e) is performed by an alcohol dehydrogenase enzyme;

wherein two or more enzymes of i)-v) are encoded by chimeric genes; and wherein said recombinant microbial host cell is capable of producing isobutanol from pyruvate through the substrate to product conversions of (a) to (e).

2. The recombinant microbial host cell of claim 1 , wherein the recombinant microbial host cell further comprises an inactivated gene thereby reducing yield loss from competing pathways for carbon flow.

3. The recombinant microbial host cell of claim 2 , wherein said inactivated gene reduces pyruvate decarboxylase activity.

4. The recombinant microbial host cell of claim 1 , wherein one or more of said substrate to product conversions is catalyzed by an enzyme that uses NADH as an electron donor.

5. The recombinant microbial host cell of claim 1 , wherein one or more of said substrate to product conversions is catalyzed by an enzyme that uses NADPH as an electron donor.

6. The recombinant microbial host cell of claim 1 , wherein the conversion of isobutyraldehyde to isobutanol is catalyzed by an NADH-dependent alcohol dehydrogenase (ADH).

7. The recombinant microbial host cell of claim 1 , wherein the conversion of isobutyraldehyde to isobutanol is catalyzed by an enzyme that is native to the recombinant microbial host cell.

8. The recombinant microbial host cell of claim 1 , wherein the host cell comprises a pyruvate decarboxylase having a preference for ketoisovalerate over pyruvate.

9. The recombinant microbial host cell of claim 1 , wherein the host cell is Escherichia.

10. The recombinant microbial host cell of claim 1 , wherein the host cell is a yeast.

11. The recombinant microbial host cell of claim 10 , wherein the yeast is Saccharomyces cerevisiae.

12. A method of producing isobutanol, comprising;

(a) providing the recombinant microbial host cell according to claim 1 ; and

(b) growing the recombinant microbial host cell under conditions whereby isobutanol is produced from pyruvate.

13. The method of claim 12 , further comprising (c) recovering the isobutanol.

14. The method of claim 12 , wherein said isobutanol is produced by batch or fed-batch fermentation.

15. The method of claim 13 , wherein said recovering is by distillation, liquid-liquid extraction, adsorption, decantation, pervaporation or combinations thereof.

16. The method of claim 13 , further comprising (d) removing solids from the fermentation medium.

17. The method of claim 16 , wherein said removing is by centrifugation, filtration or decantation.

18. The method of claim 16 , wherein said removing step (d) occurs before said recovering step (c).