Claims
- 1. An non-ribosomal peptide synthetase (NRPS) complex from Streptomyces hygroscopicus, wherein the NRPS is encoded by a nucleic acid having a nucleotide sequence as set forth in SEQ ID NO: 1, and wherein the NRPS complex is comprised of mppA and mppB.
- 2. An NRPS according to claim 1, wherein mppA is encoded by a nucleotide sequence as set forth in SEQ ID NO:3.
- 3. An NRPS according to claim 1, wherein mppB is encoded by a nucleotide sequence as set forth in SEQ ID NO:5.
- 4. An NRPS according to claim 1, wherein each of the mppA and mppB comprises a plurality of modules, each of said modules comprising an (i) adenylation domain, (ii) thiolation domain, and (iii) condensation domain.
- 5. An isolated nucleic acid having a nucleotide sequence selected from the group consisting of:
(a) a nucleotide sequence that encodes a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 21-33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 53; (b) a nucleic acid sequence that hybridizes to a nucleotide sequence encoding a polypeptide having the amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 21-33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 53, said hybridization being performed under stringent conditions; (c) a nucleic acid sequence that encodes a polypeptide at least 90% homologous to the amino acid sequence set forth in any one of SEQ ID NOs: 2, 4, 21-33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 53; and (d) an isolated nucleic acid fragment having a nucleotide sequence complementary to the nucleotide sequence of (a), (b) or (c).
- 6. An isolated nucleic acid of claim 5, wherein the nucleic acid has a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 3, 5 6-18, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.
- 7. An isolated nucleic acid according to claim 5, wherein the nucleic acid has a nucleotide sequence that is complementary to the sequence set forth in any one of SEQ ID NOs: 1, 3, 5, 6-18, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.
- 8. An isolated nucleic aid according to claim 5, wherein the nucleic acid has a nucleotide sequence that hybridizes under high stringent conditions to the sequence set forth in any of SEQ ID NOs: 1, 3, 5, 6-18, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52, or a complement thereof.
- 9. A polypeptide comprising an amino acid sequence encoded by the nucleic acid of any one of claims 5, 6, 7, or 8.
- 10. A polypeptide comprising an amino acid sequence as set forth in any one of SEQ ID NOs: 2, 4, 21-33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 53.
- 11. A chimeric nucleic acid construct comprising a nucleic acid of any one of claims 5, 6, 7, or 8, wherein said nucleic acid is operatively operatively associated with an expression control sequence.
- 12. An expression vector comprising the nucleic acid of any one of claims 5, 6, 7, or 8, wherein the nucleic acid is operatively associated with an expression control sequence.
- 13. An expression vector comprising a nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 2, wherein the nucleic acid sequence is operatively associated with an expression control sequence.
- 14. An expression vector comprising a nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4, wherein the nucleic acid sequence is operatively associated with an expression control sequence.
- 15. The expression vector according to claim 13, further comprising a second nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in SEQ ID NO: 4, wherein said second nucleic acid sequence is operatively associated with an expression control sequence.
- 16. The expression vector of claim 15, further comprising a nucleic acid sequence having a nucleotide sequence encoding a polypeptide having an amino acid sequence as set forth in any one of SEQ ID NOs: 21-33, 35, 37, 39, 41, 43, 45, 46, 49, 51, and 53 wherein said nucleic acid sequence is operatively associated with an expression control sequence.
- 17. A host cell genetically modified to express the nucleic acid of any one of claims 5, 6, 7, or 8.
- 18. A host cell genetically modified to express the nucleic acid having a nucleotide sequence as depicted in SEQ ID NO. 1.
- 19. A host cell genetically modified to express the nucleic acid of claim 5.
- 20. A host cell comprising the expression vector of claim 12.
- 21. A host cell comprising the expression vector of claim 15.
- 22. The host cell of claim 21, further comprising nucleic acid sequences having nucleotide sequences encoding polypeptides having amino acid sequences as set forth in SEQ ID NOs: 21-33, 35, 37, 39, 41, 43, 45, 47, 49, 51, and 53.
- 23. The host cell of claim 22, wherein the nucleic acids have nucleotide sequences depicted in SEQ ID NOs: 6-18, 34, 36, 38, 40, 42, 44, 46, 48, 50, and 52.
- 24. A nucleic acid having a nucleotide sequence that encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO. 21.
- 25. A nucleic acid having a nucleotide sequence that encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO. 25.
- 26. A nucleic acid having a nucleotide sequence that encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO. 28.
- 27. A nucleic acid having a nucleotide sequence that encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO. 31.
- 28. A nucleic acid having a nucleotide sequence that encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO. 32.
- 29. A nucleic acid having a nucleotide sequence that encodes a polypeptide having the amino acid sequence as set forth in SEQ ID NO. 33.
- 30. An isolated nucleic acid of claim 24, wherein the nucleic acid has a nucleotide sequence as depicted in SEQ ID NO:6.
- 31. An isolated nucleic acid of claim 25, wherein the nucleic acid has a nucleotide sequence as depicted in SEQ ID NO:10.
- 32. An isolated nucleic acid of claim 26, wherein the nucleic acid has a nucleotide sequence as depicted in SEQ ID NO:13.
- 33. An isolated nucleic acid of claim 27, wherein the nucleic acid has a nucleotide sequence as depicted in SEQ ID NO:16.
- 34. An isolated nucleic acid of claim 28, wherein the nucleic acid has a nucleotide sequence as depicted in SEQ ID NO:17.
- 35. An isolated nucleic acid of claim 29, wherein the nucleic acid has a nucleotide sequence as depicted in SEQ ID NO:18.
- 36. A method for producing NRPS, which method comprises isolating NRPS produced by the host cell of claim 22, wherein NRPS is comprised of mppA and mppB, and wherein the host cell has been cultured under conditions that provide for expression of mppA and mppB.
- 37. A method of making a peptide, the method comprising culturing the host cell of claim 22 under conditions that provide for expression of the peptide.
- 38. The method of claim 37, wherein the peptide is an antibiotic.
- 39. The method of claim 38, wherein said antibiotic is a lipoglycopeptide antibiotic with activity against Gram-positive pathogens.
- 40. The method of claim 39, wherein said antibiotic has the structure as depicted in FIG. 1.
- 41. The method of claim 36, wherein the NRPS is modified, and wherein said modified NRPS comprises addition, removal or substitution of at least one amino acid.
- 42. The method of claim 36, wherein the NRPS is modified, and wherein said modified NRPS comprises replacement of an adenylation domain.
- 43. The method of claim 41, wherein the modified NRPS comprises a modification comprising addition, removal or substitution of at least one amino acid in the group selected from SEQ ID NOs: 25, 28, 31, 32 or 33, whereby the modification results in inactivation of an O-mannosyltransferase, a phenylalanine C-methyltransferase, a first isovaleryl transferase, a second isovaleryl transferase or an arginine cyclase.
- 44. A method for determining the structural requirement of peptide activity, the method comprising:
(a) producing a modified NRPS in a host cell using the method of claim 41, (b) culturing the host cell under conditions that provide for expression of a modified peptide produced by the modified NRPS, (c) comparing the activity of the modified peptide produced by the modified NRPS with activity of a peptide produced by a non-modified NRPS; whereby a difference in the activity indicates that the modification in the modified peptide is part of the structural requirement of peptide activity.
- 45. A method for determining the structural requirement of peptide activity, the method comprising:
(a) producing a modified NRPS in a host cell using the method of claim 42, (b) culturing the host cell under conditions that provide for expression of a modified peptide produced by the modified NRPS, (c) comparing the activity of the modified peptide produced by the modified NRPS with activity of a peptide produced by a non-modified NRPS; whereby a difference in the activity indicates that the modification in the modified peptide is part of the structural requirement of peptide activity.
- 46. The method of claim 43, wherein the modified NRPS comprises a modification comprising addition, removal or substitution of at least one amino acid in the group selected from SEQ ID NOs: 25, 28, 31, 32 or 33, whereby the modification results in inactivation of an O-mannosyltransferase, a phenylalanine C-methyltransferase, a first isovaleryl transferase, a second isovaleryl transferase or an arginine cyclase.
- 47. A modified antibiotic peptide produced from a modified NRPS of claim 41.
- 48. A modified antibiotic peptide produced from a modified NRPS of claim 42.
- 49. A modified antibiotic peptide produced from a modified NRPS of claim 43.
Parent Case Info
[0001] This application claims priority from U.S. Provisional Patent Application Serial No. 60/368,713 filed on Mar. 29, 2002, herein incorporated by reference in its entirety.
Provisional Applications (1)
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Number |
Date |
Country |
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60368713 |
Mar 2002 |
US |