Leinamycin biosynthesis gene cluster and its components and their uses

Abstract

This invention provides detailed sequence analysis and characterization of the gene cluster responsible for the synthesis of leinamycin in Streptomyces atroolivaceus. The leinamycin gene cluster provides a hybrid polyketide synthase/nonribosomal peptide synthetase pathway. Elucidation of the various modules and enzymatic domains characterizing the pathway provides convenient synthetic routes for leinamycins, leinamycin analogs, and various other polyketides, peptides, and hybrid peptide-polyketide natural products.

1 . An isolated nucleic acid comprising a nucleic acid selected from the group consisting of 
 a nucleic acid encoding one or more leinamycin (inn) open reading frames (ORFs) identified in Tables 1 and 2 (ORFs −35 through −1, lnmA through lnmZ and +1 through +9);    a nucleic acid encoding a polypeptide encoded by any one or more of leinamycin (lnm) open reading frames (ORFs) identified in Tables 1 and 2 (ORFs −35 through −1, lnmA throug lnmZ and +1 through +9);    a nucleic acid comprising the nucleotide sequence of a nucleic acid amplified by polymerase chain reaction (PCR) using any one of the primer pairs identified in Table 2 and the nucleic acid of a leinamycin-producing organism as a template; and    a nucleic acid that encodes a protein comprising at least one catalytic domain selected from the group consisting of a condensation (C) domain, an adenylation (A) domain, a peptidyl carrier protein (PCP) domain, a condensation/cyclization domain (Cy), an acyl-carrier protein (ACP)-like domain, an oxidization domain (Ox), an NADH dehydrogenase domain, a methyltransferase domain, a phosphotransferase domain, a peptide synthetase domain, and an aminotransferase domain, and that specifically hybridizes to one or more leinamycin (lnm) open reading frames (ORFs) identified in Tables 1 and 2 (ORFs −35 through −1, lnmA through lnmZ, and +1 through +9) under stringent conditions.    
     
     
         2 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid comprises a nucleic acid encoding at least two open reading frames identified in Tables 1 and 2, said open reading fraes being selected from the group consisting of −35, −34, −33, −32, −31, −30, −29, −28, −27, −26, −25, −24, −23, −22, −21, −20, −19, −18, −17, −16, −15, −14, −13, −12, −11, −10, −9, −8. −7, −7, −5, −4, −3, −2, −1, lnmA, lnmB, lnmC, lnmD, lnmE, lnmF, lnmG, lmnH, lnmI, lnmJ, lmnK, lnmL, lnmM, lnmN, lmnO, InmP, lnmQ, lmnR, lnmS, lnmT, lnmU, lnmV, lnmW, lnmX, lnmY, lnmZ, =1, +2, +3, +4, +5, +6, +7, +8, and +9.  
     
     
         3 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid comprises a nucleic acid encoding at least three open reading frames identified in Tables 1 and 2, said open reading frames being selected from the group consisting of −35, −34, −33, −32, −31, −30, −29, −28, −27, −26, −25, −24, −23, −22, −21, −20, −19, −18, −17, −16, −15, −14, −13, −12, −11, −10, −9, −8. −7, −7, −5, −4, −3, −2, −1, lnmA, lnmB, lnmC, lnmD, lnmE, lnmF, lnmG, lnmH, lnmI, lnmJ, lnmK, lnmL, lnmM, lnmN, lnmO, lnmP, lnmQ, lnmR, lnmS, lnmT, lnmU, lnmV, lnmW, lnmX, lnmY, lnmZ, =1, +2, +3, +4, +5, +6, +7, +8, and +9.  
     
     
         4 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid encodes a module.  
     
     
         5 . An isolated nucleic acid of  claim 4 , comprising a nucleic acid encoding a module comprising two or more catalytic domains of a protein encoded by a nucleic acid of a leinamycin (lnm) gene cluster wherein said catalytic domains are selected from the group consisting of a condensation (C) domain, an adenylation (A) domain, a peptidyl carrier protein (PCP) domain, a condensation/cyclization domain (Cy), an acyl-carrier protein (ACP)-like domain, an oxidization domain (Ox), an enoyl reductase domain, a methyltransferase domain, a phosphotransferase domain, a peptide synthetase domain, and an aminotransferase domain.  
     
     
         6 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid comprises an open reading frame from SEQ ID NO: 1 or the complement of SEQ ID NO:1.  
     
     
         7 . The isolated nucleic acid of  claim 1 , wherein said nucleic acid has the nucleotide sequence of a nucleic acid amplified by polymerase chain reaction (PCR) using any one of the primer pairs identified in Table 2 and the nucleic acid of a leinamycin-producing organism as a template  
     
     
         8 . An isolated nucleic acid comprising a leinamycin (lnm) open reading frame (ORF) or an allelic variant thereof.  
     
     
         9 . The nucleic acid of  claim 8 , wherein said nucleic acid comprises a nucleic acid that is a single nucleotide polymorphism (SNP) of a leinamycin (lnm) open reading frame (ORF).  
     
     
         10 . An isolated gene cluster comprising open reading frames encoding polypeptides sufficient to direct the assembly of a leinamycin.  
     
     
         11 . An isolated multi-functional protein complex comprising both a polyketide synthase (PKS) and a peptide synthetase (NRPS), wherein said polyketide synthase (PKS) or said peptide synthetase (NRPS) have the amino acid sequence of a PKS or an NRPS found encoded by a nucleic acid from the leinamycin gene cluster.  
     
     
         12 . An isolated nucleic acid encoding a multi-functional protein complex comprising both a polyketide synthase (PKS) and a peptide synthetase (NRPS), wherein said polyketide synthase or said peptide synthetase, in its native state, is present in a leinamycin (lnm) gene cluster.  
     
     
         13 . An isolated polypeptide comprising a poly eptide selected from the group consisting of: 
 a catalytic domain encoded by one or more leinamycin (lnm) open reading frames (ORPs) identified in Tables 1 and 2 (ORFs −35 through −1, lnmA through lnmZ and +1 through +9);    a catalytic domain encoded by a nucleic acid having the sequence of a nucleic acid amplified by polymerase chain reaction (PCR) using any one of the primer pairs identified in Table 2; and    a module comprising two or more catalytic domains of a protein encoded by a nucleic acid of a leinamycin gene cluster.    
     
     
         14 . The polypeptide of  claim 13 , wherein said polypeptide comprises an enzymatic domain selected from the group consisting of a condensation (C) domain, an adenylation (A) domain, a peptidyl carrier protein (PCP) domain, a condensation/cyclization domain (Cy), an acyl-carrier protein (ACP)like domain, an oxidization domain (Ox), a enoyl reductase domain, a methyltransferase domain, a phosphotransferase domain, a peptide synthetase domain, and an aminotransferase domain.  
     
     
         15 . The polypeptide  claim 13 , wherein the nucleic acid of a leinamycin gene cluster comprises a nucleic acid encoding at least two open reading frames identified in Tables 1 and 2, said open reading frames being selected from the group consisting of −35, −34, −33, −32, −31, −30, −29, −28, −27, −26, −25, −24, −23, −22, −21, −20, −19, −18, −17, −16, −15, −14, −13, −12, −11, −10, −9, −8. −7, −7, −5, −4, −3, −2, −1, lnmA, lnmB, lnmC, lnmD, lnmE, lnmF, lnmG, lnmH, lnmI, lnmJ, lnmK, lnmL, lnmM, lnmN, lnmO, lnmP, lnmQ, lnmR, lnmS, lnmT, lnmU, lnmV, lnmW, lnmX, lnmY, lnmZ, =1, +2, +3, +4, +5, +6, +7, +8, and +9.  
     
     
         16 . The polypeptide  claim 13 , wherein said nucleic acid of a leinamycin gene cluster comprises a nucleic acid encoding at least three open reading frames identified in Tables 1 and 2, said open reading frames being selected from the group consisting of −35, −34, −33, −32, −31, −30, −29, −28, −27, −26, −25, −24, −23, −22, −21, −20, −19, −18, −17, −16, −15, −14, −13, −12, −11, −10, −9, −8. −7, −7, −5, −4, −3, −2, −1, lnmA, lnmB, lnmC, lnmD, lnmE, lnmF, lnmG, lnmH, lnmI, lnmJ, lnmK, lnmL, lnmM, lnmN, lnmO, lnmP, lnmQ, lnmR, lnmS, lnmT, lnmU, lnmV, lnmW, lnmX, lnmY, lnmZ, =1, +2, +3, +4, +5, +6, +7, +8, and +9.  
     
     
         17 . The polypeptide of  claim 13 , wherein said polypeptide comprises a module comprising two or more catalytic domains of a protein encoded by a nucleic acid of a leinamycin gene cluster wherein said catalytic domains are selected from the group consisting of a condensation (C) domain, an adenylation (A) domain, a peptidyl carrier protein (PCP) domain, a condensation/cyclization domain (Cy), an acyl-carrier protein (ACP)-like domain, an oxidization domain (Ox), an enoyl reductase domain (ER), a methyltransferase domain, a phosphotransferase domain, a peptide synthetase domain, and an aminotransferase domain.  
     
     
         18 . An isolated polypeptide comprising a module wherein said module is specifically bound by an antibody that specifically binds to a leinamycin (lnm) module.  
     
     
         19 . The polypeptide of  claim 18 , wherein said polypeptide is specifically bound by an antibody that specifically binds to a polypeptide encoded by a leinamycin open reading frame.  
     
     
         20 . An expression vector comprising a nucleic acid of any one of claims  1  through  12 .  
     
     
         21 . A host cell transformed with an expression vector of  claim 20 .  
     
     
         22 . The host cell of  claim 21 , wherein said cell is transformed with an exogenous nucleic acid comprising a gene cluster encoding polypeptides sufficient to direct the assembly of a leinamycin or leinamycin analog.  
     
     
         23 . The cell of  claim 21 , wherein said cell is a bacterial cell.  
     
     
         24 . The cell of  claim 23 , wherein said cell is a  Streptomyces  cell.  
     
     
         25 . The cell of  claim 21 , wherein said cell is a eukaryotic cell.  
     
     
         26 . The cell of  claim 21 , wherein said cell is an insect cell.  
     
     
         27 . A method of chemically modifying a molecule, said method comprising contacting a molecule that is a substrate for a polypeptide encoded by one or more leinamycin biosynthesis gene cluster open reading frames with a polypeptide encoded by one or more leinamycin biosynthesis bene cluster open reading frames, whereby said polypeptide chemically modifies said molecule.  
     
     
         28 . The method of  claim 27 , wherein said method comprising contacting said molecule with at least two different polypeptides encoded by leinamycin (lnm) gene cluster open reading frames.  
     
     
         29 . The method of  claim 27 , wherein said method comprising contacting said molecule with at least three different polypeptides encoded by leinamycin (lnm) gene cluster open reading frames.  
     
     
         30 . The method of  claim 27 , wherein said contacting is in a host cell.  
     
     
         31 . The method of  claim 30 , wherein said host cell is a bacterium.  
     
     
         32 . The method of  claim 27 , wherein said contacting ex vivo.  
     
     
         33 . The method of  claim 27 , wherein said molecule is an endogenous metabolite produced by said host cell.  
     
     
         34 . The method of  claim 27 , wherein said molecule is an exogenous supplied metabolite.  
     
     
         35 . The method of  claim 27 , wherein said host cell is a eukaryotic cell.  
     
     
         36 . The method of  claim 35 , wherein said eukaryotic cell is selected from the group consisting of a mammnalian cell, a yeast cell, a plant cell, a fungal cell, and an insect cell.  
     
     
         37 . The method of  claim 27 , wherein said molecule is an amino acid and said polypeptide is a peptide synthetase.  
     
     
         38 . The method of  claim 27 , wherein said polypeptide is an amino transferase.  
     
     
         39 . A cell that overexpresses leinamycin.  
     
     
         40 . The cell of  claim 39 , wherein said cell overexpresses a polypeptide encoded by leinamycin open reading frame lnmL.  
     
     
         41 . A cell that produces leinamycin, wherein one or more proteins that synthesize said leinamycin are encoded by one or more heterologous nucleic acids.  
     
     
         42 . The cell of  claim 41 , wherein said heterologous nucleic acids comprise at least three open reading frames identified in Tables 1 and 2, said open reading frames being selected from the group consisting of −35, −34, −33, −32, −31, −30, −29, −28, −27, −26, −25, −24, −23, −22, −21, −20, −19, −18, −17, −16, −15, −14, −13, −12, −11, −10, −9, −8, −7, −7, −5, −4, −3, −2, −1, lnmA, lnmB, lnmC, lnmD, lnmE, lnmF, lnmG, lnmH, lnmI, lnmJ, lnmK, lnmL, lnmM, lnmN, lnmO, lnmP, lnmQ, lnmR, lnmS, lnmT, lnmU, lnmV, lnmW, lnmX, lnmY, lnmZ, +1, +2, +3, +4, +5, +6, +7, +8, and +9  
     
     
         43 . A method of coupling a first amino acid to a second amino acid, said method comprising contacting the first and second amino acid with a recombinantly expressed leinamycin nonribosomal peptide synthetase (NRPS).  
     
     
         44 . The method of  claim 49 , wherein said NRPS is selected from the group consisting of NRPS-1, and NRPS-2.  
     
     
         45 . The method of  claim 49 , wherein said contacting is in a host cell.  
     
     
         46 . A method of coupling a first fatty acid to a second fatty acid, said method comprising contacting the first and second fatty acids with a recombinantly expressed leinamycin polyketide synthase (PKS).  
     
     
         47 . The method of  claim 46 , wherein said PKS is selected from the group consisting of lnm PKS-1, PKS-2, PKS-3, PKS-4, PKS-5, and PKS-6.  
     
     
         48 . The method of  claim 46 , said contacting is in a host cell.  
     
     
         49 . A method of producing a leinamycin or leinamycin analog, said method comprising: 
 providing a cell transformed with an exogenous nucleic acid comprising a leinamycin gene cluster encoding polypeptides sufficient to direct the assembly of said leinamycin or leinamycin analog;    culturing the cell under conditions permitting the biosynthesis of leinamycin or leinamycin analog; and    isolating said leinamycin or leinamycin analog from said cell.    
     
     
         50 . A method of producing a leinamycin analog, said method comprising: 
 providing a cell comprising a leinamycin gene cluster;    transfecting the cell with a nucleic acid that alters the leinamycin gene cluster through homologous recombination so that the gene cluster encodes a biosynthetic pathway that synthesizes,said leinamycin analog;    culturing the cell under conditions permitting the biosynthesis of the leinamycin analog; and    isolating the leinamycin analog from the cell.    
     
     
         51 . An isolated nucleic acid comprising a nucleic acid encoding a phosphopantetheinyl transferase said nucleic acid encoding a phosphopantetheinyl transferase being selected from the group consisting of: 
 a nucleic acid encoding the protein comprising the amino acid sequence encoded by lmp of  FIG. 5 ;    a nucleic acid encoding a polypeptide having phosphopantetheinyl transferase activity where said nucleic acid specifically hybridizes to the nucleic acid having the sequence encoded by lmp of  FIG. 5  under stringent conditions.    
     
     
         52 . The nucleic acid of  claim 51 , wherein said nucleic acid comprises the sequence of lmp in  FIG. 5 .  
     
     
         53 . The nucleic acid of  claim 51 , wherein said nucleic acid comprises a vector.  
     
     
         54 . A polypeptide comprising a phosphopantetheinyl transferase encoded by a nucleic acid of  claim 51 .  
     
     
         55 . A vector comprising the nucleic acid of  claim 51 .  
     
     
         56 . A cell transfected with the vector of  claim 55 .  
     
     
         57 . A method of converting an apo-carrier protein to a holo-carrier protein comprising reacting said apo-carrier protein with a recombinant phosphopantetheinyl transferase encoded by the nucleic acid of  claim 51  and coenzyme A thereby producing a holo-carrier protein.  
     
     
         58 . A cell comprising a modified leinamycin gene cluster nucleic acid, said cell producing elevated amounts of leinamycin as compared to the wild type cell.  
     
     
         59 . The cell of  claim 58 , wherein said cell overexpresses a resistance gene from the leinamycin gene cluster.  
     
     
         60 . The cell of  claim 59 , wherein said resistance gene is a gene listed in Table 2.  
     
     
         61 . An antibody that specifically binds to a polypeptide encoded by an lnm open reading frame identified in Table 2.  
     
     
         62 . The antibody of  claim 61 , wherein said antibody is a single chain antibody.