Recombinant host cells comprising phosphoketolases

1. A recombinant host cell comprising:

(i) at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity;

(ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and

(iii) a pyruvate-utilizing biosynthetic pathway, wherein said pyruvate-utilizing biosynthetic pathway is an isobutanol biosynthetic pathway comprising the substrate to product conversions:

(a) pyruvate to acetolactate;

(b) acetolactate to 2,3-dihydroxyisovalerate;

(c) 2,3-dihydroxyisovalerate to 2-ketoisovalerate;

(d) 2-ketoisovalerate to isobutyraldehyde; and

(e) isobutyraldehyde to isobutanol; and

wherein said recombinant host cell produces isobutanol.

2. The recombinant host cell of claim 1 , wherein said host cell further comprises:

(iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

3. The recombinant host cell of claim 1 , wherein said host cell has a reduced requirement for an exogenous two-carbon substrate for its growth in culture compared to a recombinant eukaryotic host cell comprising (i) and not (ii).

4. The recombinant host cell of claim 1 , wherein said host cell has improved growth as compared to a host cell comprising (i) and not (ii) in culture media that is not supplemented with an exogenous two-carbon substrate.

5. The recombinant host cell of claim 4 , wherein said exogenous two-carbon substrate is ethanol or acetate.

6. The recombinant host cell of claim 1 , wherein said endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is PDC1, PDC5, PDC6, or combinations thereof; and said host cell is S. cerevisiae.

7. The recombinant host cell of claim 1 , wherein said heterologous polynucleotide encoding a polypeptide having phosphoketolase activity is xpk1 from Lactobacillus plantarum , xpkA from Lactobacillus pentosus MD363 or 6-phosphate phosphoketolase from B. lactis.

8. The recombinant host cell of claim 1 wherein said polypeptide having phosphoketolase activity comprises at least 85% identity to SEQ ID NO: 481 or an active fragment thereof.

9. The recombinant host cell of claim 2 , wherein said heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity is EutD from Lactobacillus plantarum or phosphotransacetylase from Bacillus subtilis.

10. The recombinant host cell of claim 2 , wherein said phosphotransacetylase polypeptide comprises at least 85% identity to SEQ ID NO:1472 or an active fragment thereof.

11. The recombinant host cell of claim 1 , wherein said host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula , or Saccharomyces.

12. The recombinant host cell of claim 1 , wherein said host cell is Saccharomyces cerevisiae.

13. A recombinant host cell comprising:

(i) at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity;

(ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and

(iii) a pyruvate-utilizing biosynthetic pathway, wherein said pyruvate-utilizing biosynthetic pathway is a 2-butanone biosynthetic pathway comprising the substrate to product conversions:

(i) pyruvate to acetolactate;

(ii) acetolactate to acetoin;

(iii) acetoin to 2,3-butanediol;

(iv) 2,3-butanediol to 2-butanone; and

wherein said recombinant host cell produces 2-butanone.

14. A recombinant host cell comprising:

(i) at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity;

(ii) a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity; and

(iii) a pyruvate-utilizing biosynthetic pathway, wherein said pyruvate-utilizing biosynthetic pathway is a 2-butanol biosynthetic pathway comprising the substrate to product conversions:

(i) pyruvate to acetolactate;

(ii) acetolactate to acetoin;

(iii) acetoin to 2,3-butanediol;

(iv) 2,3-butanediol to 2-butanone;

(v) 2-butanone to 2-butanol; and

wherein said recombinant host cell produces 2-butanol.

15. The recombinant host cell of claim 1 wherein the phosphoketolase matches the Profile HMM given in Table 6 with an E value of less than 7.5E-242.

16. The recombinant host cell of claim 1 wherein the phosphoketolase matches the Profile HMMs given in Tables 6, 7, 8, and 9 with E values of less than 7.5E-242, 1.1E-124, 2.1E-49, and 7.8E-37, respectively.

17. The recombinant host cell of claim 1 further comprising a phosphotransacetylase which matches the Profile HMM given in Table 14 with an E value of less than 5E-34.

18. The recombinant host cell of claim 13 , wherein said host cell further comprises:

(iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

19. The recombinant host cell of claim 13 , wherein said host cell has a reduced requirement for an exogenous two-carbon substrate for its growth in culture compared to a recombinant eukaryotic host cell comprising (i) and not (ii).

20. The recombinant host cell of claim 13 , wherein said host cell has improved growth as compared to a host cell comprising (i) and not (ii) in culture media that is not supplemented with an exogenous two-carbon substrate.

21. The recombinant host cell of claim 20 , wherein said exogenous two-carbon substrate is ethanol or acetate.

22. The recombinant host cell of claim 13 , wherein said endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is PDC1, PDC5, PDC6, or combinations thereof; and said host cell is S. cerevisiae.

23. The recombinant host cell of claim 13 , wherein said heterologous polynucleotide encoding a polypeptide having phosphoketolase activity is xpk1 from Lactobacillus plantarum , xpkA from Lactobacillus pentosus MD363 or 6-phosphate phosphoketolase from B. lactis.

24. The recombinant host cell of claim 13 , wherein said polypeptide having phosphoketolase activity comprises at least 85% identity to SEQ ID NO: 481 or an active fragment thereof.

25. The recombinant host cell of claim 18 , wherein said heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity is EutD from Lactobacillus plantarum or phosphotransacetylase from Bacillus subtilis.

26. The recombinant host cell of claim 18 , wherein said phosphotransacetylase polypeptide comprises at least 85% identity to SEQ ID NO:1472 or an active fragment thereof.

27. The recombinant host cell of claim 13 , wherein said host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula , or Saccharomyces.

28. The recombinant host cell of claim 13 , wherein said host cell is Saccharomyces cerevisiae.

29. The recombinant host cell of claim 14 , wherein said host cell further comprises:

(iii) a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

30. The recombinant host cell of claim 14 , wherein said host cell has a reduced requirement for an exogenous two-carbon substrate for its growth in culture compared to a recombinant eukaryotic host cell comprising (i) and not (ii).

31. The recombinant host cell of claim 4 , wherein said host cell has improved growth as compared to a host cell comprising (i) and not (ii) in culture media that is not supplemented with an exogenous two-carbon substrate.

32. The recombinant host cell of claim 31 , wherein said exogenous two-carbon substrate is ethanol or acetate.

33. The recombinant host cell of claim 14 , wherein said endogenous gene encoding a polypeptide having pyruvate decarboxylase activity is PDC1, PDCS, PDC6, or combinations thereof; and said host cell is S. cerevisiae.

34. The recombinant host cell of claim 14 , wherein said heterologous polynucleotide encoding a polypeptide having phosphoketolase activity is xpk1 from Lactobacillus plantarum , xpkA from Lactobacillus pentosus MD363 or 6-phosphate phosphoketolase from B. lactis.

35. The recombinant host cell of claim 14 , wherein said polypeptide having phosphoketolase activity comprises at least 85% identity to SEQ ID NO: 481 or an active fragment thereof.

36. The recombinant host cell of claim 29 , wherein said heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity is EutD from Lactobacillus plantarum or phosphotransacetylase from Bacillus subtilis.

37. The recombinant host cell of claim 29 , wherein said polypeptide having phosphotransacetylase activity comprises at least 85% identity to SEQ ID NO:1472 or an active fragment thereof.

38. The recombinant host cell of claim 14 , wherein said host cell is a member of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Serratia, Erwinia, Klebsiella, Shigella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Schizosaccharomyces, Kluyveromyces, Yarrowia, Pichia, Candida, Hansenula , or Saccharomyces.

39. The recombinant host cell of claim 14 , wherein said host cell is Saccharomyces cerevisiae.

40. A method for the production of 2-butanol, or 2-butanone comprising growing the recombinant host cell of claim 13 or 14 under conditions wherein the product is produced and optionally recovering the product.

41. A method for the production of isobutanol comprising growing the recombinant host cell of claim 1 under conditions wherein the product is produced and optionally recovering the product.

42. A method of producing the recombinant host cell of claim 1 comprising:

transforming a host cell comprising at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity, with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity.

43. The method of claim 42 , wherein said method further comprises:

transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

44. A method of improving the growth of a recombinant host cell comprising at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity, comprising:

transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity,

wherein the recombinant host cell comprising the heterologous polynucleotide encoding a polypeptide having phosphoketolase activity has improved growth as compared to a control recombinant host cell without the heterologous polynucleotide encoding a polypeptide having phosphoketolase activity.

45. The method of claim 44 , wherein said method further comprises:

transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.

46. The method of claim 44 , further comprising growing the recombinant host cell in media containing limited carbon substrate.

47. A method of reducing the requirement for an exogenous two-carbon substrate for the growth of a recombinant host cell comprising at least one genetic modification in an endogenous gene encoding a polypeptide having pyruvate decarboxylase activity, wherein the at least one genetic modification reduces or eliminates pyruvate decarboxylase activity, comprising: transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphoketolase activity, wherein the recombinant host cell comprising the heterologous polynucleotide encoding a polypeptide having phosphoketolase activity has a reduced requirement for an exogenous two-carbon substrate as compared to a control recombinant host cell without the heterologous polynucleotide encoding a polypeptide having phosphoketolase activity.

48. The method of claim 47 , wherein said method further comprises:

transforming said recombinant host cell with a heterologous polynucleotide encoding a polypeptide having phosphotransacetylase activity.