METHODS, COMPOSITIONS AND SYSTEMS FOR BIOSYNTHETIC BIO-PRODUCTION OF 1,4-BUTANEDIOL

• US 20110190513A1
• Filed: 07/08/2009
• Published: 08/04/2011
• Est. Priority Date: 07/08/2008
• Status: Abandoned Application

• Current Assignee: OPX Biotechnologies, Inc. (Cargill, Inc. (Agricultural Services Division))
• Original Assignee: OPX Biotechnologies, Inc. (Cargill, Inc. (Agricultural Services Division))
• Inventors: Lynch, Michael D.
• Application Number:
  US13/002,941
• Publication Number:
  US 20110190513A1
• Time in Patent Office: Days
• US Class Current: 549/295
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: C12N 9/88 Lyases (4.)
• CPC Class Codes: C12N 9/90 Isomerases (5.)
• CPC Class Codes: C12P 7/16 Butanols
• CPC Class Codes: C12P 7/18 polyhydric
• CPC Class Codes: Y02E 50/10 Biofuels, e.g. bio-diesel

Abstract

Three biosynthetic pathways are disclosed for microorganism bio-production of 1,4-Butanediol from various carbon sources. Exemplary methods are provided. The recombinant microorganisms comprising any of these 1,4-Butanediol biosynthesis pathways may also comprise genetic modifications directed to improved tolerance for 1,4-Butanediol.

Claims

1-31. -31. (canceled)

32. A recombinant microorganism comprising nucleic acid sequences encoding polypeptides that convert 4-hydroxybutyrate (“

4-HB”

) to 4-hydroxybutanal and 4-hydroxybutanal to 1,4-butanediol (“

1,4-BDO”

), thereby biosynthesizing 1,4-BDO from carbon sources provided to the recombinant microorganism.

33. The recombinant microorganism of claim 32 comprising nucleic acid sequences encoding polypeptides for at least one of the following pathways:

a. a pathway that converts citrate to cis-aconitate to D-isocitrate to alpha ketoglutarate to succinate semialdehyde to the 4-HB;

b. a pathway that converts acetyl-CoA to acetoacetyl-CoA to 3-hydroxybutryryl-CoA to crotonyl-CoA to vinylacetyl-CoA to 4-hydroxybutyryl-CoA to the 4-HB;

or

c. a pathway that converts succinate to succinate semialdehyde (optionally including via succinyl-CoA) to the 4-HB;

thereby providing 4-HB.

53. An industrial-scale microbial bioreactor system comprising:

(a) a bioreactor vessel;

(b) a carbon source;

(c) a recombinant microorganism of claim 32; and

(d) a media;

wherein the carbon source is optionally selected from the group consisting of sucrose, glucose, xylose, cellulose or hemicelluloses; and

wherein the media is optionally a minimal media; and

further wherein said minimal media optionally comprises M9 minimal media, potassium sulfate minimal media, yeast synthetic minimal media and variations thereof.

34. A recombinant microorganism comprising a biosynthetic pathway with aconitase, isocitrate dehydrogenase, and alpha ketoglutarate decarboxylase enzymatic activities.

35. The recombinant microorganism of claim 34 additionally comprising 4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase, and 1,3 propanediol dehydrogenase enzymatic activities.

36. The recombinant microorganism of claim 34, additionally comprising a biosynthetic pathway with acetyl-coA acetyltransferase, beta-hydroxybutyryl-coA dehydrogenase, crotonase, vinylacetyl-coA-isomerase, 4-hydroxybutyryl-coA dehydratase, and 4-hydroxybutyrate coA transferase enzymatic activities.

37. The recombinant microorganism of claim 34, additionally comprising a biosynthetic pathway with succinate semialdehyde dehydrogenase, and optionally with succinyl-coA synthetase, enzymatic activities.

44. A method of biosynthesis of a polymer chemistry intermediate comprising providing in a bioreactor vessel a recombinant microorganism of claim 34, a carbon source, and a media, and conducting a bio-production event under suitable conditions and for a suitable time to obtain a measurable quantity of the intermediate.

45. The method of claim 44, wherein the intermediate is 1,4-BDO.

46. A method of making a polyester, a polyurethane, butyrolactone, γ

-butyrolactone, or tetrahydrofuran comprising converting the 1,4-BDO of claim 45 to a polyester, a polyurethane, butyrolactone, γ

-butyrolactone, or tetrahydrofuran.

38. A recombinant microorganism comprising a biosynthetic pathway with acetyl-coA acetyltransferase, beta-hydroxybutyryl-coA dehydrogenase, crotonase, vinylacetyl-coA-isomerase, 4-hydroxybutyryl-coA dehydratase, and 4-hydroxybutyrate coA transferase enzymatic activities.

39. The recombinant microorganism of claim 38, additionally comprising 4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase, and 1,3 propanediol dehydrogenase enzymatic activities.

40. A recombinant microorganism of claim 38, additionally comprising a biosynthetic pathway with succinate semialdehyde dehydrogenase, and optionally with succinyl-coA synthetase, enzymatic activities.

41. A recombinant microorganism of claim 40, comprising modifications in nucleic acids encoding respective polypeptides of the biosynthetic pathways, and resulting in biosynthesis of 3-hydroxybutyryl-coA.

47. A method of biosynthesis of a polymer chemistry intermediate comprising providing in a bioreactor vessel a recombinant microorganism of claim 38, a carbon source, and a media, and conducting a bio-production event under suitable conditions and for a suitable time to obtain a measurable quantity of the intermediate.

48. The method of claim 47, wherein the intermediate is 1,4-BDO.

49. A method of making a polyester, a polyurethane, butyrolactone, γ

-butyrolactone, or tetrahydrofuran comprising converting the 1,4-BDO of claim 48 to a polyester, a polyurethane, butyrolactone, γ

-butyrolactone, or tetrahydrofuran.

42. A recombinant microorganism comprising a biosynthetic pathway with succinate semialdehyde dehydrogenase, and optionally with succinyl-coA synthetase, enzymatic activities.

43. The recombinant microorganism of claim 42 additionally comprising 4-hydroxybutyrate dehydrogenase, aldehyde dehydrogenase, and 1,3 propanediol dehydrogenase enzymatic activities.

50. A method of biosynthesis of a polymer chemistry intermediate comprising providing in a bioreactor vessel a recombinant microorganism of claim 42, a carbon source, and a media, and conducting a bio-production event under suitable conditions and for a suitable time to obtain a measurable quantity of the intermediate.

51. The method of claim 50, wherein the intermediate is 1,4-BDO.

52. A method of making a polyester, a polyurethane, butyrolactone, γ

-butyrolactone, or tetrahydrofuran comprising converting the 1,4-BDO of claim 51 to a polyester, a polyurethane, butyrolactone, γ

-butyrolactone, or tetrahydrofuran.