Recombinant Streptomyces hygroscopicus host cells that produce 17-desmethylrapamycin

Abstract

Recombinant host cells that produce rapamycin analogues are constructed by deleting or modifying rapamycin biosynthetic gene cluster genes and are useful in the production of compounds used as antifungals, anticancers, immunosuppressants, and neurotrophins.

1 . A recombinant  Streptomyces hygroscopicus  host cell that produces 16-desmethyl-27-desmethoxyrapamycin.  
     
     
         2 . The compound 16-desmethyl-27-desmethoxyrapamycin in substantially pure form.  
     
     
         3 . A recombinant  Streptomyces hygroscopicus  host cell that produces a compound selected from the group consisting of 17,18-dihydrorapamycin, 19,20-dihydrorapamycin, 21,22-dihydrorapamycin, 17,18,19,20-tetrahydrorapamycin, 17,18,21,22-tetrahydrorapamycin, 19,20,21,22-tetrahydrorapamycin, 17,18,19,20,21,22-hexahydrorapamycin, 16-demethyl-17,18-dihydrorapamycin, 16-demethyl-19,20-dihydrorapamycin, 16-demethyl-21,22-dihydrorapamycin, 16-demethyl-17,18,19,20-tetrahydrorapamycin, 16-demethyl-17,18,21,22-tetrahydrorapamycin, 16-demethyl-19,20,21,22-tetrahydrorapamycin, 16-demethyl-17,18,19,20,21,22-hexahydrorapamycin, 17-desmethylrapamycin, 23-desmethylrapamycin, 17,23-didesmethylrapamycin, 17-desmethyl-17,18-dihydrorapamycin, 17-desmethyl-19,20-dihydrorapamycin, 17-desmethyl-21,22-dihydrorapamycin, 17-desmethyl-17,18,19,20-tetrahydrorapamycin, 17-desmethyl-17,18.21,22-tetrahydrorapamycin, 17-desmethyl-19,20,21,22-tetrahydrorapamycin, 17-desmethyl-17,18,19,20,21,22-hexahydrorapamycin, 23-desmethyl-17,18-dihydrorapamycin, 23-desmethyl-19,20-dihydrorapamycin, 23-desmethyl-21,22-dihydrorapamycin, 23-desmethyl-17,18,19,20-tetrahydrorapamycin, 23-desmethyl-17,18,21,22-tetrahydrorapamycin, 23-desmethyl-19,20,21,22-tetrahydrorapamycin, 23-desmethyl-17,18,19,20,21,22-hexahydrorapamycin, 17,23-didesmethyl-17,18-dihydrorapamycin, 17,23-didesmethyl-19,20-dihydrorapamycin, 17,23-didesmethyl-21,22-dihydrorapamycin, 17,23-didesmethyl-17,18,19,20-tetrahydrorapamycin, 17,23-didesmethyl-17,18,21,22-tetrahydrorapamycin, 17,23-didesmethyl-19,20,21,22-tetrahydrorapamycin, 17,23-didesmethyl-17,18,19,20,21,22-hexahydrorapamycin, 19-methylrapamycin, 19,20-del-rapamycin, 18-hydroxyrapamycin, 18-ketorapamycin, and 18-saturated-rapamycin.  
     
     
         4 . A compound selected from the group consisting of 17,18-dihydrorapamycin, 19,20-dihydrorapamycin, 21,22-dihydrorapamycin, 17,18,19,20-tetrahydrorapamycin, 17,18,21,22-tetrahydrorapamycin, 19,20,21,22-tetrahydrorapamycin, 17,18,19,20,21,22-hexahydrorapamycin, 16-demethyl-17,18-dihydrorapamycin, 16-demethyl-19,20-dihydrorapamycin, 16-demethyl-21,22-dihydrorapamycin, 16-demethyl-17,18,19,20-tetrahydrorapamycin, 16-demethyl-17,18,21,22-tetrahydrorapamycin, 16-demethyl-19,20,21,22-tetrahydrorapamycin, 16-demethyl-17,18,19,20,21,22-hexahydrorapamycin, 17-desmethylrapamycin, 23-desmethylrapamycin, 17,23-didesmethylrapamycin, 17-desmethyl-17,18-dihydrorapamycin, 17-desmethyl-19,20-dihydrorapamycin, 17-desmethyl-21,22-dihydrorapamycin, 17-desmethyl-17,18,19,20-tetrahydrorapamycin, 17-desmethyl-17,18.21,22-tetrahydrorapamycin, 17-desmethyl-19,20,21,22-tetrahydrorapamycin, 17-desmethyl-17,18,19,20,21,22-hexahydrorapamycin, 23-desmethyl-17,18-dihydrorapamycin, 23-desmethyl-19,20-dihydrorapamycin, 23-desmethyl-21,22-dihydrorapamycin, 23-desmethyl-17,18,19,20-tetrahydrorapamycin, 23-desmethyl-17,18,21,22-tetrahydrorapamycin, 23-desmethyl-19,20,21,22-tetrahydrorapamycin, 23-desmethyl-17,18,19,20,21,22-hexahydrorapamycin, 17,23-didesmethyl-17,18-dihydrorapamycin, 17,23-didesmethyl-19,20-dihydrorapamycin, 17,23-didesmethyl-21,22-dihydrorapamycin, 17,23-didesmethyl-17,18,19,20-tetrahydrorapamycin, 17,23-didesmethyl-17,18,21,22-tetrahydrorapamycin, 17,23-didesmethyl-19,20,21,22-tetrahydrorapamycin, 17,23-didesmethyl-17,18,19,20,21,22-hexahydrorapamycin, 19-methylrapamycin, 19,20-del-rapamycin, 18-hydroxyrapamycin, 18-ketorapamycin, and 18-saturated-rapamycin, in substantially pure form.  
     
     
         5 . A recombinant  Streptomyces hygroscopicus  host cell that expresses a hybrid PKS composed of at least a portion of a rapamycin PKS and at least a portion of a heterologous PKS.  
     
     
         6 . A recombinant  Streptomyces hygroscopicus  host cell that does not express at least one rapamycin modification enzyme but does produce a rapamycin analogue.  
     
     
         7 . A recombinant  Streptomyces hygroscopicus  host cell that expresses a PKS composed of only a portion of a rapamycin PKS.  
     
     
         8 . A recombinant  Streptomyces hygroscopicus  host cell that does not express at least one rapamycin modification enzyme but does produce a rapamycin analogue produced by a hybrid PKS composed of at least a portion of a rapamycin PKS and at least a portion of a heterologous PKS.  
     
     
         9 . A recombinant  Streptomyces hygroscopicus  host cell that does not express at least one rapamycin modification enzyme but does produce a rapamycin analogue produced by a PKS consisting essentially of only a portion of a rapamycin PKS.  
     
     
         10 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 6  in which rapI gene, rapJ, rapM, or rapQ has been deleted or inactivated.  
     
     
         11 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 10  in which rapI gene, rapJ, rapM, or rapQ has been deleted or inactivated.  
     
     
         12 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 11  in which rapN and rapO have been deleted or inactivated.  
     
     
         13 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 12  in which rapN, rapO and rapM have been deleted or inactivated.  
     
     
         14 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 13  in which rapM, rapN, rapO, and rapQ have been deleted or inactivated.  
     
     
         15 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 14  in which rapL, rapM, rapN, rapO, and rapQ have been deleted or inactivated.  
     
     
         16 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 6  that produces 16-desmethyl-27-desmethoxyrapamycin.  
     
     
         17 . The recombinant  Streptomyces hygroscopicus  host cell of  claim 6  that expresses a rapamycin PKS derivative in which at least one domain of one module of the is deleted or inactivated, wherein said cells does not comprise a rapamycin PKS derivative that contains a heterologous PKS domain, module, or protein.  
     
     
         18 . A method of making a rapamycin analog comprising culturing a host cell of  claim 6  under conditions where the analog is produced.  
     
     
         19 . The method of  claim 18  wherein the rapamycin analog is 16-desmethyl-27-desmethoxyrapamycin.  
     
     
         20 . 16-desmethyl-27-desmethoxyrapamycin produced by a method comprising culturing a host cell of  claim 16  under conditions where 16-desmethyl-27-desmethoxyrapamycin is produced.