Claims
- 1. An engineered microorganism, wherein the microorganism produces at least 200 μg α-lipoic acid (ALA) per g dry cell weight of the engineered microorganism.
- 2. The engineered microorganism of claim 1, wherein the microorganism comprises an exogenous nucleic acid encoding a lipoic acid synthase polypeptide.
- 3. The engineered microorganism of claim 2, wherein the microorganism further comprises an exogenous nucleic acid encoding at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoic acid synthase polypeptide.
- 4. The engineered microorganism of claim 3, wherein the at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoic acid synthase polypeptide is an iron-sulfur cluster S (IscS) polypeptide.
- 5. The engineered microorganism of claim 4, wherein the IscS polypeptide is cysteine sulfinic desulfinase (Csd).
- 6. The engineered microorganism of claim 3, wherein the at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoic acid synthase polypeptide is an iron-sulfur cluster U (IscU) polypeptide.
- 7. The engineered microorganism of claim 3, wherein the at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoic acid synthase polypeptide is an IscS polypeptide and an IscU polypeptide.
- 8. The engineered microorganism of claim 1, wherein the microorganism comprises an exogenous lipoic acid synthase polypeptide, an exogenous IscS polypeptide, and an exogenous IscU polypeptide.
- 9. The engineered microorganism of claim 1, wherein the microorganism is a prokaryote.
- 10. The engineered microorganism of claim 9, wherein the prokaryote is of the Escherichia genus.
- 11. The engineered microorganism of claim 10, wherein the prokaryote is Escherichia coli.
- 12. The engineered microorganism of claim 2, wherein the microorganism is lacking an endogenous transcriptional regulator ORF.
- 13. The engineered microorganism of claim 12, transcriptional regulator ORF is as IscR.
- 14. A method of making ALA, comprising extracting ALA from the engineered microorganism of claim 1.
- 15. The method of claim 14, wherein the engineered microorganism comprises an exogenous lipoic acid synthase polypeptide, an IscS polypeptide, or an exogenous IscU polypeptide.
- 16. The method of claim 14, wherein the engineered microorganism comprises an exogenous lipoic acid synthase polypeptide, an IscS polypeptide, and an exogenous IscU polypeptide.
- 17. A composition comprising ALA produced by the engineered microorganism of claim 1.
- 18. A nutraceutical comprising ALA produced by the engineered microorganism of claim 1.
- 19. A pharmaceutical comprising ALA produced by the engineered microorganism of claim 1.
- 20. An isolated nucleic acid comprising a first expression control element operably linked to an nucleic acid encoding a lipoic acid synthase polypeptide and a second expression control element operably linked to a nucleic acid encoding an IscS polypeptide and/or an IscU polypeptide.
- 21. The isolated nucleic acid of claim 20, wherein the IscS polypeptide is Csd.
- 22. A vector comprising the isolated nucleic acid of claim 20.
- 23. A cell comprising the isolated nucleic acid of claim 20.
- 24. An engineered microorganism, wherein the microorganism secretes at least a 2-fold greater amount of ALA than an amount of ALA found intracellularly.
- 25. The engineered microorganism of claim 24, wherein the microorganism secretes at least a 5-fold greater amount of ALA than an amount of ALA found intracellularly.
- 26. The engineered microorganism of claim 24, wherein the microorganism secretes at least a 10-fold greater amount of ALA than an amount of ALA found intracellularly.
- 27. The engineered microorganism of claim 24, wherein the microorganism secretes at least 8.7 μg ALA per g dry cell weight and contains less than 1.5 μg ALA per g dry cell weight intracellularly.
- 28. The engineered microorganism of claim 24, wherein the microorganism is a eukaryote.
- 29. The engineered microorganism of claim 28, wherein the eukaryote is a member of the genus Saccharomyces.
- 30. The engineered microorganism of claim 29, wherein the eukaryote is S. cerevisiae.
- 31. The engineered microorganism of claim 24, wherein the microorganism comprises an exogenous nucleic acid encoding a lipoate synthase polypeptide.
- 32. The engineered microorganism of claim 31, wherein the microorganism further comprises an exogenous nucleic acid encoding at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoate synthase polypeptide.
- 33. The engineered microorganism of claim 32, wherein the at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoate synthase polypeptide is an Nfs1 polypeptide.
- 34. The engineered microorganism of claim 24, wherein the microorganism comprises an exogenous lipoate synthase polypeptide and an exogenous Nfs1 polypeptide.
- 35. A nucleic acid construct comprising a first expression control element operably linked to a nucleic acid encoding a lipoate synthase polypeptide and a second expression control element operably linked to a nucleic acid encoding an Nfs1 polypeptide.
- 36. A method of making ALA, comprising purifying ALA secreted by the engineered microorganism of claim 24.
- 37. A composition comprising ALA produced by the engineered microorganism of claim 24.
- 38. A nutraceutical comprising ALA produced by the engineered microorganism of claim 24.
- 39. A pharmaceutical comprising ALA produced by the engineered microorganism of claim 24.
- 40. A method of producing ALA comprising culturing an engineered microorganism expressing an exogenous nucleic acid encoding a lipoic acid synthase polypeptide under conditions which permit the microorganism to produce ALA.
- 41. The method of claim 40, further comprising expressing an exogenous nucleic acid encoding at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoic acid synthase polypeptide.
- 42. The method of claim 41, wherein the at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoic acid synthase polypeptide is an IscS polypeptide and/or an IscU polypeptide.
- 43. The method of claim 41, wherein the at least one polypeptide that assembles or regenerates an Fe—S cluster of the lipoic acid synthase polypeptide is an Nfs1 polypeptide.
- 44. The method of claim 40, wherein the microorganism produces at least 200 μg ALA per g dry cell weight.
- 45. The method of claim 40, wherein the microorganism secretes at least a 2-fold greater amount of ALA than an amount of ALA found intracellularly.
CROSS REFERENCE TO RELATED APPLICATION
[0001] This claims the benefit of U.S. Provisional Application No. 60/285,276 filed Apr. 20, 2001.
PCT Information
Filing Document |
Filing Date |
Country |
Kind |
PCT/US02/12458 |
4/19/2002 |
WO |
|
Provisional Applications (1)
|
Number |
Date |
Country |
|
60285276 |
Apr 2001 |
US |