Compositions and methods relating to the daptomycin biosynthetic gene cluster

• US 20050027113A1
• Filed: 07/31/2002
• Published: 02/03/2005
• Est. Priority Date: 08/06/2001
• Status: Abandoned Application

• Current Assignee: Cubist Pharmaceuticals, Inc. (Merck & Co., Inc.)
• Original Assignee: Cubist Pharmaceuticals, Inc. (Merck & Co., Inc.)
• Inventors: Coeffet-LeGal, Marie Francoise, Miao, Vivian Pak Woon, Baltz, Richard H., Brian, Paul
• Application Number:
  US10/211,028
• Publication Number:
  US 20050027113A1
• Time in Patent Office: Days
• US Class Current: 536/23.200
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes

Abstract

The invention provides nucleic acid molecules comprising all or a part of a daptomycin biosynthetic gene cluster. The daptomycin biosynthetic gene cluster may be derived from Streptomyces, preferably from S. roseosporus. The invention also provides other nucleic acid molecules from S. roseosporus. The invention further provides polypeptides encoded by the nucleic acid molecules, antibodies that specifically bind to the polypeptides, and methods of using the nucleic acid molecules, polypeptides and antibodies to produce daptomycin and other compounds.

Claims

1. An isolated nucleic acid molecule comprising a nucleic acid sequence encoding a daptomycin non-ribosomal peptide synthetase (NRPS) or subunit thereof from Streptomyces roseosporus, wherein the nucleic acid molecule encodes DptBC and wherein said nucleic acid molecule is not pRHB159.

2. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule encodes a polypeptide comprising amino acid sequence SEQ ID NO:

11.

3. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule is dptBC or wherein the nucleic acid molecule comprises nucleic acid sequence SEQ ID NO:

12.

4. The nucleic acid molecule according to claim 1, wherein the nucleic acid molecule comprises a nucleic acid sequence from an S. roseosporus nucleic acid sequence from BAC clone B12:

03A05.

22. A vector comprising the nucleic acid molecule according to any one of claims 1-21.

23. The vector according to claim 22, wherein the vector comprises expression control sequences controlling the transcription of the nucleic acid molecule.

24. The vector according to claim 23 wherein the expression control sequences control the expression of the nucleic acid molecule in a prokaryotic cell.

25. A host cell comprising the nucleic acid molecule according to any one of claims 1-21.

26. A host cell comprising the vector according to any one of claims 22-24.

27. A method for producing DptBC, or a domain, module or part thereof, comprising the step of culturing the host cell according to claims 25 or 26 under conditions in which the polypeptide is produced, optionally comprising the step of isolating the polypeptide.

29. An isolated polypeptide that is encoded by the nucleic acid molecule according to any one of claims 1-13.

37. A method for determining if a sample contains a nucleic acid molecule encoding a daptomycin NRPS or a daptomycin NRPS subunit, comprising the steps of

(a) providing a nucleic acid molecule according to any one of claims 1-21;

(b) contacting the nucleic acid molecule with the sample under selective hybridization conditions; and

(c) determining if the nucleic acid molecule selectively hybridized to a nucleic acid molecule in the sample.

5. An isolated nucleic acid molecule that encodes a daptomycin NRPS or subunit thereof, wherein the isolated nucleic acid molecule selectively hybridizes to a reference nucleic acid molecule that encodes a daptomycin NRPS or subunit thereof, wherein the reference nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:

a) the nucleic acid sequence of dptBC;

(b) a nucleic acid sequence encoding the amino acid sequence of DptBC;

(c) a nucleic acid sequence encoding the amino acid sequence SEQ ID NO;

11; and

(d) the nucleic acid sequence SEQ ID NO;

12;

wherein said nucleic acid molecule is not pRHB159.

6. The isolated nucleic acid molecule according to claim 5, wherein the nucleic acid molecule hybridizes under conditions selected from the group consisting of low stringency conditions, moderate stringency conditions and high stringency conditions.

7. An isolated nucleic acid molecule that encodes a daptomycin NRPS or subunit thereof, wherein the isolated nucleic acid molecule comprises a nucleic acid sequence that has at least 50% sequence identity to a nucleic acid sequence selected from the group consisting of:

(a) the nucleic acid sequence of dptBC;

(b) a nucleic acid sequence encoding the amino acid sequence of DptBC;

(c) a nucleic acid sequence encoding the amino acid sequence SEQ ID NO;

11; and

(d) the nucleic acid sequence SEQ ID NO;

12;

wherein said nucleic acid molecule is not pRHB159.

8. The nucleic acid molecule according to claim 7, wherein the homologous molecule exhibits at least 60% sequence identity to the nucleic acid sequence of any one of (a) to (d).

9. The nucleic acid molecule according to claim 8, wherein the sequence identity is at least 70%.

10. The nucleic acid molecule according to claim 9, wherein the sequence identity is at least 80%.

11. The nucleic acid molecule according to claim 10, wherein the sequence identity is at least 90%.

12. The nucleic acid molecule according to claim 11, wherein the sequence identity is at least 95%.

13. An isolated nucleic acid molecule that encodes a daptomycin NRPS or subunit thereof, wherein the isolated nucleic acid molecule is an allelic variant of a nucleic acid molecule that comprises a nucleic acid sequence selected from the group consisting of:

(a) the nucleic acid sequence of dptBC;

(b) a nucleic acid sequence encoding the amino acid sequence of DptBC;

(c) a nucleic acid sequence encoding the amino acid sequence SEQ ID NO;

11; and

(d) the nucleic acid sequence SEQ ID NO;

12.

14. An isolated nucleic acid molecule comprising a part of a nucleic acid sequence, wherein said part is at least 14 nucleotides, selected from the group consisting of:

(a) a nucleic acid sequence encoding the amino acid sequence of a polypeptide comprising amino acids 5003 to 5007 Of SEQ ID NO;

11; and

(b) a nucleic acid sequence comprising nucleotides 68350 to 68364 of SEQ ID NO;

12;

wherein said nucleic acid molecule is not pRHB159.

15. The isolated nucleic acid molecule according to claim 14, wherein said nucleic acid molecule encodes at least one domain from a daptomycin NRPS.

16. The isolated nucleic acid molecule according to claim 14, wherein said nucleic acid molecule encodes at least one module from a daptomycin NRPS.

17. The nucleic acid molecule according to claim 14, wherein the part comprises at least 17 nucleotides of the nucleic acid sequence.

18. The nucleic acid molecule according to claim 17, wherein the part comprises at least 20 nucleotides of the nucleic acid sequence.

19. The nucleic acid molecule according to claim 18, wherein the part comprises at least 25 nucleotides of the nucleic acid sequence.

20. The nucleic acid molecule according to claim 19, wherein the part comprises at least 50 nucleotides of the nucleic acid sequence.

21. The nucleic acid molecule according to any one of claims 14-20 that is an oligonucleotide from 14 to 60 nucleotides in length.

30. An isolated polypeptide that is encoded by the nucleic acid molecule according to any one of claims 14-21.

28. An isolated polypeptide comprising an amino acid sequence selected from the group consisting of

(a) the amino acid sequence encoded by the nucleic acid sequence of dptBC;

(b) the amino acid sequence of DptBC;

(c) the amino acid sequence of SEQ ID NO;

11; and

(d) the amino acid sequence encoded by the nucleic acid sequence SEQ ID NO;

12.

31. An antibody that selectively binds to the polypeptide according to any one of claims 28-30.

32. The antibody according to claim 31 that is an intact immunoglobulin;

an antigen-binding portion thereof that is Fab, Fab′

, F(ab′

)₂, Fv, dAb or a CDR fragment;

a single-chain antibody;

a chimeric antibody;

a diabody;

or a polypeptide comprising at least a portion of the immunoglobulin sufficient to confer specific antigen binding to the polypeptide.

33. The antibody according to claim 32, wherein the antibody is a neutralizing antibody.

34. The antibody according to claim 32, wherein the antibody is an activating antibody.

35. The antibody according to claim 32, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

36. A method for preparing an antibody that selectively binds to the polypeptide according to any one of claims 28-30, comprising the steps of

(a) immunizing a non-human animal with the polypeptide;

and

(b) isolating the antibody

38. A method to produce daptomycin comprising the steps of

(a) introducing a nucleic acid molecule comprising SEQ ID NO:

1 in a host cell; and

(b) culturing the host cell under conditions in which daptomycin is produced.

39. The method according to claim 38, wherein the nucleic acid molecule is derived from Streptomyces.

40. The method according to claim 38 wherein the nucleic acid molecule is derived from S. roseosporus.

41. The method according to claim 40, wherein the nucleic acid molecule comprises the entire daptomycin biosynthetic gene cluster.

42. The method according to claim 38, wherein the nucleic acid molecule encodes a peptide comprising DptA, DptBC or DptD.

43. The method according to claim 42, wherein the nucleic acid molecule encodes a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:

9, SEQ ID NO;

11 and SEQ ID NO;

7.

44. The method according to claim 38 wherein the nucleic acid molecule conprises a nucleic acid sequence selected from the group consisting of dptA, dptBC and dptD, or wherein the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO:

10, SEQ ID NO;

12 and SEQ ID NO;

3.

45. The method according to claim 38, wherein the nucleic acid molecule comprises a nucleic acid sequence from an S. roseosporus nucleic acid sequence from BAC clone B12:

03A05.

46. The method according to claim 38, wherein the nucleic acid molecule selectively hybridizes to a reference nucleic acid molecule that encodes a daptomycin gene cluster or a portion thereof, wherein the reference nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:

(a) a nucleic acid sequence selected from the group consisting of dptA, dptBC and dptD;

(b) a nucleic acid sequence encoding the amino acid sequence of a polypeptide selected from the group consisting of DptA, DptBC and DptD;

(c) a nucleic acid sequence encoding the amino acid sequence of a polypeptide selected from the group consisting of SEQ ID NO;

9, SEQ ID NO;

11 and SEQ ID NO;

7;

(d) a nucleic acid sequence selected from the group consisting of SEQ ID NO;

10, SEQ ID NO;

12 and SEQ ID NO;

3;

(e) a nucleic acid sequence from an S. roseosporus nucleic acid sequence from BAC clone B12;

03A05.

47. The method according to claim 38, further comprising the step of isolating the daptomycin.

48. A method to increase the production of daptomycin by a cell comprising the steps of

(a) providing a host cell that expresses daptomycin;

(b) introducing a nucleic acid molecule into a neutral site of a chromosome of said host cell, wherein the introduction of the nucleic acid molecule results in increased production of daptomycin by a cell compared to the cell without the nucleic acid molecule; and

(c) culturing the host cell under conditions in which daptomycin is produced;

wherein the nucleic acid molecule comprises a nucleic acid sequence selected from the group consistng of

(i) the nucleic acid sequence of dptBC;

(ii) a nucleic acid sequence encoding the amino acid sequence of DptBC;

(iii) a nucleic acid sequence encoding the amino acid sequence SEQ ID NO;

11; and

(iv) the nucleic acid sequence SEQ ID NO;

12.

49. The method according to claim 48, wherein the host cell is S. roseosporus or S. lividans comprising the daptomycin biosynthetic gene cluster.

50. A nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS:

24, 54, 74, 94, 98 and 100, or encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOS;

93, 97 and 99;

wherein said nucleic acid molecule is not pRHB160, pRHB166, pRHB613, pRHB614, or pRHB680.

51. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:

93, 97, and 99;

or encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NOS;

24, 54, 74, 94 98 and 100.

54. An antibody that binds to the polypeptide according to either of claims 51 or 53.

52. A nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOS:

105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 and 135;

or encoding a polypeptide having an amino acid sequence selected from the group consisting of 104, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136;

wherein said nucleic acid molecule is not pRHB174.

53. A polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:

104, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134 and 136;

or encoded by a nucleic acid molecule selected from the group consisting of SEQ ID NOS;

102, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133 and 135.

55. A method for producing a lipopeptide or peptide core thereof, comprising the steps of

(a) providing a cell comprising an NRPS gene cluster other than the daptomycin biosynthetic gene cluster in a cell;

(b) introducing into the cell at least one gene that encodes all or a part of the NRPS from the daptomycin biosynthetic gene cluster; and

(c) culturing the cell under conditions in which the lipopeptide or peptide core thereof is produced.

57. The method according to either of claims 55 or 56, wherein said daptomycin biosynthetic gene cluster comprises a naturally-occurring nucleotide sequence of the daptomycin biosynthetic gene cluster.

58. The method according to either of claims 55 or 56, wherein said daptomycin biosynthetic gene cluster comprises a modified nucleotide sequence compared to the naturally-occurring nucleotide sequence of the daptomycin biosynthetic gene cluster.

59. The method according to claim 58, wherein said modified nucleotide sequence comprises a modification or replacement of one or more modules of the daptomycin biosynthetic gene cluster that alters one or more amino acids incorporated by the NRPS encoded by the daptomycin biosynthetic gene cluster compared to the amino acids encoded by the NRPS of the naturally-occurring daptomycin biosynthetic gene cluster.

60. The method according to either of claims 55 or 56, wherein said NRPS gene cluster other than the daptomycin biosynthetic gene cluster comprises a naturally-occurring nucleotide sequence of the NRPS gene cluster.

61. The method according to either of claims 55 or 56, wherein said NRPS gene cluster other than the daptomycin biosynthetic gene cluster comprises a modified nucleotide sequence compared to the naturally-occurring nucleotide sequence of the NRPS gene cluster.

62. The method according to claim 61, wherein said modified nucleotide sequence comprises a modification or replacement of one or more modules of the NRPS gene cluster that alters one or more amino acids incorporated by the NRPS gene cluster compared to the amino acids encoded by the NRPS of the naturally-occurring NRPS gene cluster.

63. The method according to either of claims 55 or 56, wherein said daptomycin biosynthetic gene cluster comprises a naturally-occurring nucleotide sequence of the daptomycin biosynthetic gene cluster and said NRPS gene cluster other than the daptomycin biosynthetic gene cluster comprise a naturally-occurring nucleotide sequence of the NRPS gene cluster.

64. The method according to either of claims 55 or 56, wherein said daptomycin biosynthetic gene cluster comprises a modified nucleotide sequence compared to the naturally-occurring nucleotide sequence of the daptomycin biosynthetic gene cluster and said NRPS gene cluster other than the daptomycin biosynthetic gene cluster comprises a modified nucleotide sequence compared to the naturally-occurring nucleotide sequence of the NRPS gene cluster.

65. The method according to claim 64, wherein said modified nucleotide sequence of said daptomycin biosynthetic gene cluster and of said NRPS gene cluster other than the daptomycin biosynthetic gene cluster comprises a modification or replacement of one or more modules of the daptomycin biosynthetic gene cluster or the NRPS gene cluster that alters one or more amino acids incorporated by the NRPS gene cluster compared to the amino acids encoded by the NRPS of the naturally-occurring gene cluster.

66. The method according to either of claims 55 or 56, further comprising the step of introducing into the cell at least one more gene that encodes all or a part of an NRPS from an NRPS gene cluster other than the daptomycin biosynthetic gene cluster.

67. The method according to either of claims 55 or 56, further comprising the step of introducing into the cell at least one more gene that encodes all or a part of an NRPS from the daptomycin biosynthetic gene cluster.

68. The method according to either of claims 55 or 56, further comprising the step of isolating the lipopeptide or peptide core thereof.

69. The cell produced by the method according to either of claims 55 or 56.

70. The lipopeptide or peptide core thereof produced by the method according to either of claims 55 or 56.

56. A method for producing a lipopeptide or peptide core thereof, comprising the steps of

(a) providing a cell comprising a daptomycin biosynthetic gene cluster in a cell;

(b) introducing into the cell at least one other gene that encodes all or a part of an NRPS from an NRPS gene cluster other than the daptomycin biosynthetic gene cluster; and

(c) culturing the cell under conditions in which the lipopeptide or peptide core thereof is produced.

71. A method for producing a lipopeptide or peptide core thereof, comprising the steps of

(a) providing a cell comprising a daptomycin biosynthetic gene cluster in a cell;

(b) modifying or exchanging one or more modules of the daptomycin biosynthetic gene cluster to alter at least one amino acid directed for incorporation by the gene cluster;

(c) introducing into the cell at least one other gene that encodes all or a part of an NRPS from the daptomycin biosynthetic gene cluster or another NRPS gene cluster; and

(d) culturing the cell under conditions in which the lipopeptide or peptide core thereof is produced.

72. A method for producing a lipopeptide or peptide core thereof, comprising the steps of

(a) providing a cell comprising an NRPS gene cluster in a cell;

(b) introducing a daptomycin biosynthetic gene cluster in a cell;

(c) modifying or exchanging one or more modules of the daptomycin biosynthetic gene cluster to alter at least one amino acid directed for incorporation by the gene cluster; and

(d) culturing the host cell under conditions in which the lipopeptide or peptide core thereof is produced.

73. A method for producing a lipopeptide or peptide core thereof, comprising the steps of

(a) providing a cell comprising an NRPS gene cluster in a cell;

(b) providing a daptomycin biosynthetic gene cluster in a cell;

(c) modifying or exchanging one or more modules of the daptomycin biosynthetic gene cluster to alter at least one amino acid directed for incorporation by the gene cluster;

(d) introducing the modified daptomycin biosynthetic gene cluster into the cell; and

(e) culturing the host cell under conditions in which the lipopeptide or peptide core thereof is produced.