Non-ribosomal peptide synthetases and associated biosynthetic genes

Abstract

The present invention describes the identification of novel non-ribosomal peptide synthetases and associated biosynthetic genes from Streptomyces hygroscopicus . The present invention further provides methods for generating novel compounds, such as antibiotics, from these synthetases and associated genes.

What is claimed:  
     
         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.