Novel spinosyn-producing polyketide synthases

Abstract

The invention provides, biologically active spinosyns, hybrid spinosyn polyketide synthases capable of functioning in Saccharopolyspora spinosa to produce the spinosyns, and methods of controlling insects using the spinosyns.

1 . A compound of the formula (I)  
       
         
           
           
               
               
           
         
       
       wherein 
 R1 is hydrogen, methyl, or ethyl;  
 R2 is hydrogen, methyl, or ethyl;  
 R3 is hydrogen,  
                     
 R4 is methyl or ethyl, either of which may be substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano;  
 R4 is an alpha-branched C 3 -C 5  alkyl group, C 3 -C 8  cycloalkyl group, or C 3 -C 8  cycloalkenyl group, any of which may be substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano; or  
 R4 is a 3-6 membered heterocyclic group that contains O or S, that is saturated or fully or partially unsaturated, and that may be substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano;  
 R5 is hydrogen or methyl;  
 R6 is hydrogen or methyl;  
 R7 is hydrogen or methyl;  
 R8 is hydrogen, methyl, or ethyl;  
 R9 is hydrogen, methyl, or ethyl;  
 or a 5,6-dihydro derivative of a compound of formula I, provided that the compound has at least one of the following features:  
 a) R4 is methyl or ethyl substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano; or  
 b) R4 is an alpha-branched C 3 -C 5  alkyl group, C 3 -C 8  cycloalkyl group, or C 3 -C 8  cycloalkenyl group, any of which may be substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano; or  
 c) R4 is a 3-6 membered heterocyclic group that contains O or S, that is saturated or fully or partially unsaturated, and that may be substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano; or  
 d) R1 or R2 is ethyl; or  
 e) R8 is methyl or ethyl; or  
 f) R9 is methyl or ethyl.  
 
     
     
         2 . A compound of  claim 1  wherein R1 is ethyl.  
     
     
         3 . A compound of  claim 1  wherein R2 is ethyl.  
     
     
         4 . A compound of  claim 1  wherein R4 is 
 methyl or ethyl substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano; or  
 an alpha-branched C 3 -C 5  alkyl group, C 3 -C 8  cycloalkyl group, or C 3 -C 8  cycloalkenyl group, any of which may be substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano; or  
 a 3-6 membered heterocyclic group that contains O or S, that is saturated or fully or partially unsaturated, and that may be substituted with one or more groups selected from halo, hydroxy, C 1 -C 4  alkyl, C 1 -C 4  alkoxy, C 1 -C 4  alkylthio, or cyano.  
 
     
     
         5 . A compound of  claim 4  wherein R4 is the residue of a carboxylic acid useable as a substrate by the ery or ave loading modules.  
     
     
         6 . A compound of  claim 4  wherein R4 is n-propyl, iso-propyl, cyclopropyl, methylcyclopropyl, sec-butyl, cyclobutyl, methylthio, or furyl.  
     
     
         7 . A compound selected from the group consisting of: 
 21-desethyl-21-cyclopropyl spinosyn A;    21-desethyl-21-cyclopropyl spinosyn D;    21-desethyl-21-cyclobutyl spinosyn A;    21-desethyl-21-cyclobutyl spinosyn D;    21-desethyl-21-methylthiomethyl spinosyn A;    21-desethyl-21-methylthiomethyl spinosyn D;    21-desethyl-21-cyanomethyl spinosyn A;    5,6-dihydro-21-desethyl-21-cyclobutyl spinosyn A;    21-desethyl-21-isopropyl spinosyn A;    21-desethyl-21-isopropyl spinosyn D;    21-desethyl-21-sec-butyl spinosyn A;    21-desethyl-21-sec-butyl spinosyn D;    21-desethyl-21-methylcyclopropyl spinosyn A;    21-desethyl-21-methylcyclopropyl spinosyn D;    21-desethyl-21-(3-furyl) spinosyn A;    21-desethyl-21-(3-furyl) spinosyn D;    21-desethyl-21-cyclopropyl spinosyn A 17-pseudoaglycone;    21-desethyl-21-cyclopropyl spinosyn D 17-pseudoaglycone;    21-desethyl-21-cyclobutyl spinosyn A 17-pseudoaglycone;    21-desethyl-21-cyclobutyl spinosyn D 17-pseudoaglycone;    16-desmethyl spinosyn D;    16-desmethyl spinosyn D 17-pseudoaglycone;    21-desethyl-21-n-propyl spinosyn A;    6-ethyl spinosyn A;    6-ethyl-21-desethyl-21-n-propyl spinosyn A;    16-desmethyl-16-ethyl spinosyn A;    16-desmethyl-16-ethyl spinosyn D; and    21-desethyl-21-n-propyl spinosyn D.    
     
     
         8 . A hybrid spinosyn polyketide synthase that is capable of functioning in  Saccharopolyspora spinosa  to produce a biologically active spinosyn, said hybrid polyketide synthase comprising a heterologous loading module operatively associated with a plurality of  Saccharopolyspora spinosa  extender modules.  
     
     
         9 . A hybrid spinosyn polyketide synthase of  claim 8  comprising a loading module of SEQ ID NO:8.  
     
     
         10 . A hybrid spinosyn polyketide synthase of  claim 8  comprising a loading module of SEQ ID NO:11.  
     
     
         11 . A hybrid spinosyn polyketide synthase that is capable of functioning in  Saccharopolyspora spinosa  to produce a biologically active 16-ethyl spinosyn compound, said hybrid polyketide synthase being the product produced by spinosyn biosynthetic DNA modified so that the DNA for the AT domain of module 3 in the spinosyn PKS is replaced with DNA for an AT domain that normally incorporates ethyl malonyl-CoA.  
     
     
         12 . A hybrid spinosyn polyketide synthase that is capable of functioning in  Saccharopolyspora spinosa  to produce a biologically active 18-ethyl spinosyn compound, said hybrid polyketide synthase being the product produced by spinosyn biosynthetic DNA modified so that the DNA for the AT domain of module 2 in the spinosyn PKS is replaced with DNA for an AT domain that normally incorporates ethyl malonyl-CoA.  
     
     
         13 . A hybrid spinosyn polyketide synthase that is capable of functioning in  Saccharopolyspora spinosa  to produce a biologically active 20-ethyl spinosyn compound, said hybrid polyketide synthase being the product produced by spinosyn biosynthetic DNA modified so that the DNA for the AT domain of module 1 in the spinosyn PKS is replaced with DNA for an AT domain that normally incorporates ethyl malonyl-CoA.  
     
     
         14 . A hybrid spinosyn polyketide synthase that is capable of functioning in  Saccharopolyspora spinosa  to produce a biologically active 16-ethyl spinosyn compound, 20-ethyl spinosyn compound, or 18-ethyl spinosyn compound, said hybrid polyketide synthase being the product produced by spinosyn biosynthetic DNA modified so that the DNA for the AT domain of module 3, module 2, or module 1 in the spinosyn PKS is replaced with DNA for the AT domain of module 5 in the tylosin PKS.  
     
     
         15 . A hybrid spinosyn polyketide synthase of  claim 14  wherein said AT domain is encoded by the DNA of SEQ ID NO:26.  
     
     
         16 . A hybrid spinosyn polyketide synthase that is capable of functioning in  Saccharopolyspora spinosa  to produce a biologically active 18-methyl spinosyn compound, said hybrid polyketide synthase being the product produced by spinosyn biosynthetic DNA modified so that the DNA for the AT domain of module 2 in the spinosyn PKS is replaced with DNA for an AT domain that normally incorporates methyl malonyl-CoA.  
     
     
         17 . A hybrid spinosyn polyketide synthase that is capable of functioning in  Saccharopolyspora spinosa  to produce a biologically active 20-methyl spinosyn compound, said hybrid polyketide synthase being the product produced by spinosyn biosynthetic DNA modified so that the DNA for the AT domain of module 1 in the spinosyn PKS is replaced with DNA for an AT domain that normally incorporates methyl malonyl-CoA.  
     
     
         18 . A process for producing a 6-ethyl spinosyn compound, a 21-desethyl-21-n-propyl spinosyn compound, or a 6-ethyl-21-desethyl-21-n-propyl spinosyn compound which comprises culturing a host organism that coexpresses crotonyl-CoA reductase with the spinosyn biosynthetic pathway.  
     
     
         19 . The process of  claim 18  wherein the host organism is an  S. spinosa  strain that has been transformed with DNA encoding the  S. cinnamonensis  crotonyl-CoA reductase.  
     
     
         20 . An  S. spinosa  strain that has been transformed with DNA encoding the  S. cinnamonensis  crotonyl-CoA reductase.  
     
     
         21 . A biologically pure culture of  Saccharopolyspora spinosa  selected from 
 NRRL 30539,    NRRL 30540,    NRRL 30541, and    NRRL 30542.    
     
     
         22 . A method of controlling pests which comprises delivering to a pest an effective amount of a compound of  claim 1 .  
     
     
         23 . A pesticide composition comprising an effective amount of a compound of  claim 1  as active ingredient in combination with an appropriate diluent or carrier