Combinatorial polyketide libraries produced using a modular PKS gene cluster as scaffold

Abstract

Combinatorial libraries of polyketides can be obtained by suitable manipulation of a host modular polyketide synthase gene cluster such as that which encodes the PKS for erythromycin. The combinatorial library is useful as a source of pharmaceutically active compounds.

1 . A method for modifying the acyltransferase domain in a modular polyketide synthase (PKS) comprising replacing a first region encoding a first acyltransferase domain of a first PKS with a second region encoding a second acyltransferase domain from a second PKS.  
     
     
         2 . The method of  claim 1  wherein the first region and second region only encode an acyltransferase domain.  
     
     
         3 . The method of  claim 1  wherein the first PKS is from  Saccharopolyspora erythraea.    
     
     
         4 . The method of  claim 1  wherein the second PKS is from  Saccharopolyspora erythraea.    
     
     
         5 . The method of  claim 1  wherein the first PKS and/or second PKS is selected from the group consisting of rapamycin, avermectin, FK-506, and tylosin.  
     
     
         6 . The method of  claim 1  wherein the first and/or second PKS is selected from Streptomyces.  
     
     
         7 . The method of  claim 6  wherein the Streptomyces is  Streptomyces hygroscopicus.    
     
     
         8 . The method of  claim 1  wherein the replacement of the acyltransferase domain is conducted by in vivo recombination.  
     
     
         9 . The method of  claim 8  wherein the recombination is via homologous sequences framing the replacement region in a donor plasmid and a receptor region in a recipient plasmid.  
     
     
         10 . The method of  claim 1  wherein the replacement of the acyltransferase domain is conducted in vitro using restriction enzymes.  
     
     
         11 . A vector comprising the first or second PKS of  claim 1  or the PKS produced by the method of  claim 1 .  
     
     
         12 . A host cell transformed with the vector of  claim 11 .  
     
     
         13 . The host cell of  claim 12  wherein said cell is a bacterial cell.  
     
     
         14 . The host cell of  claim 12  wherein said bacterial cell is  E. coli.    
     
     
         15 . The host cell of  claim 12  wherein said cell is a polyketide-producing organism.  
     
     
         16 . The host cell of  claim 12  wherein said polyketide-producing organism is selected from the group consisting of a Streptomyces species and a Saccharopolyspora species.  
     
     
         17 . A method for altering the substrate specificity of a polyketide synthase in a first polyketide-producing microorganism comprising the steps of: 
 (a) isolating a first and second genomic DNA segment, each comprising a polyketide synthase wherein said first genomic DNA segment is from said first polyketide-producing microorganism and said second genomic DNA segment is from said first polyketide-producing microorganism or a second polyketide-producing microorganism;    (b) identifying one or more discrete fragments of said first genomic DNA segment, each of which encodes an acyltransferase domain;    (c) identifying one or more discrete fragments of said second genomic DNA segment, each of which encodes a related domain to said acyltransferase domain of said first genomic DNA segment; and    (d) transforming a cell of said first polyketide-producing microorganism with one or more of said fragments from step (c) under conditions suitable for the occurrence of a homologous recombination event, leading to the replacement of one or more of said fragments from said first genomic DNA segment with one or more of said fragments from step (c).    
     
     
         18 . The method of  claim 17  wherein said first polyketide-producing microorganism is  Saccharopolyspora erythraea.    
     
     
         19 . The method of  claim 17  wherein said second polyketide-producing microorganism is  Saccharopolyspora erythraea.    
     
     
         20 . The method of  claim 17  wherein said acyltransferase domain is selected from a module of DEBS or a module of rapamycin synthase.  
     
     
         21 . The method of  claim 17  wherein said first polyketide-producing microorganism is Streptomyces.  
     
     
         22 . The method of  claim 21  wherein said Streptomyces is selected from the group consisting of  Streptomyces antibioticus, Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyces caelestis, Streptomyces tsukubaensis, Streptomyces fradiae, Streptomyces platensis, Streptomyces violaceoniger, Streptomyces ambofaciens, Streptomyces griseoplanus, and Streptomyces venezuelae.    
     
     
         23 . The method of  claim 22  wherein said Streptomyces is selected from the group consisting of  Streptomyces caelestis  and  Streptomyces venezuelae.    
     
     
         24 . The method of  claim 17  wherein said second polyketide-producing microorganism is Streptomyces.  
     
     
         25 . The method of  claim 24  wherein said Streptomyces is selected from the group consisting of  Streptomyces antibioticus, Streptomyces mycarofaciens, Streptomyces avermitilis, Streptomyces hygroscopicus, Streptomyces caelestis, Streptomyces tsukubaensis, Streptomyces fradiae, Streptomyces platensis, Streptomyces violaceoniger, Streptomyces ambofaciens , and  Streptomyces venezuelae.    
     
     
         26 . The method of  claim 25  wherein said Streptomyces is selected from the group consisting of  Streptontyces caelestis, Streptomyces hygroscopicus , and  Streptomyces venezuelae .