Biosynethetic gene cluster for jerangolids

Abstract

Domains of jerangolid polyketide synthase and modification enzymes and polynucleotides encoding them are provided. Methods to prepare jerangolid in pharmaceutically useful quantities are described, as are methods to prepare jerangolid analogs and other polyketides using the polynucleotides encoding jerangolid synthase domains or modifying enzymes.

1 . A purified or recombinant nucleic acid comprising a nucleotide sequence that encodes at least one polypeptide required for the biosynthesis of jerangolid, wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of nucleotides 1-67323 of SEQ ID NO:1, under conditions of hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C. 
     
     
         2 . A purified or recombinant nucleic acid a nucleotide sequence that encodes at least one module of the jerangolid polyketide synthase, wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of nucleotides that encode modules of the jerangolid PKS as listed in Table 1. 
     
     
         3 . A purified or recombinant nucleic acid according to  claim 1 , wherein said polypeptide comprises a β-ketoacylsynthase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of β-ketoacylsynthase domains as listed in Table 1, under conditions of hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C. 
     
     
         4 . A purified or recombinant nucleic acid according to  claim 1 , wherein said polypeptide comprises an acyltransferase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of acyltransferase domains as listed in Table 1, under conditions of hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C. 
     
     
         5 . A purified or recombinant nucleic acid according to  claim 1 , wherein said polypeptide comprises a β-ketoreductase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of β-ketoreductase domains as listed in Table 1, under conditions of hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C. 
     
     
         6 . A purified or recombinant nucleic acid according to  claim 1 , wherein said polypeptide comprises a dehydratase domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of dehydratase domains as listed in Table 1, under conditions of hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C. 
     
     
         7 . A purified or recombinant nucleic acid according to  claim 1 , wherein said polypeptide comprises an enoylreductase domain and wherein the complement of said nucleotide sequence hybridizes to enoylreductase domains as listed in Table 1, under conditions of hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C. 
     
     
         8 . A purified or recombinant nucleic acid according to  claim 1 , wherein said polypeptide comprises an acyl carrier protein domain and wherein the complement of said nucleotide sequence hybridizes to a sequence selected from the group consisting of acyl carrier protein domains as listed in Table 1, under conditions of hybridization at 65° C. for 36 hours and washing 3 times at high stringency with 0.1×SSC and 0.5% SDS for 20 minutes at 65° C. 
     
     
         9 . A purified or recombinant polypeptide involved in the biosynthesis of an jerangolid, wherein said polypeptide has an amino acid sequence that can be encoded by a nucleic acid sequence of  claim 1 . 
     
     
         10 . The polypeptide of  claim 9  that can be encoded by the gene jerA. 
     
     
         11 . The polypeptide of  claim 9  that can be encoded by the gene jerB. 
     
     
         12 . The polypeptide of  claim 9  that can be encoded by the gene jerC. 
     
     
         13 . The polypeptide of  claim 9  that can encoded by the gene jerD. 
     
     
         14 . The polypeptide of  claim 9  that can be encoded by the gene jerE. 
     
     
         15 . The polypeptide of  claim 9  that can be encoded by the gene jerF. 
     
     
         16 . A method of making an jerangolid or jerangolid analog, said method comprising expressing at least one recombinant gene of  claim 1  in a host cell capable of producing polyketides.