Polyketides and their synthesis

Abstract

A polyketide synthase (“PKS”) of Type I is a complex multienzyme including a loading domain linked to a multiplicity of extension domains. The first extension module receives an acyl starter unit from the loading domain and each extension module adds a further ketide unit which may undergo processing (e.g. reduction). We have found that the Ksq domain possessed by some PKS's has decarboxylating activity, e.g. generating (substituted) acyl from (substituted) malonyl. The CLF domain of type II PKS's has similar activity. By inserting loading modules including such domains into PKS's not normally possessing them it is possible to control the starter units used.

1 . A system for use in producing a polyketide having substantially exclusively a desired starter unit by providing a PKS multienzyme which comprises a loading module and a plurality of extension modules, wherein said loading module is adapted to load an optionally substituted malonyl and then to effect decarboxylation of the loaded residue to provide a corresponding optionally substituted acetyl residue for transfer to an adjacent one of said extension modules, and wherein at least one of the extension modules is not naturally associated with a loading module that effects decarboxylation; with the proviso that the target polyketide is not a 14-membered macrolide having a 13-methyl group due to incorporation of an (unsubstituted) acetate starter unit.  
     
     
         2 . A system according to  claim 1  wherein said adjacent extension module to which the acetate starter is transferred is not naturally associated with a loading module that effects decarboxylation.  
     
     
         3 . A system according to  claim 1  wherein the decarboxylating functionality of the loading module is provided by a ketosynthase-type domain having a glutamine residue in the active site or other residue other than cysteine.  
     
     
         4 . A system according to  claim 1  wherein the decarboxylating functionality of the loading module is provided by a CLF-type domain.  
     
     
         5 . A system according to  claim 1  wherein the loading module's loading functionality is provided by an acyltransferase-type domain having an arginine residue in the active site.  
     
     
         6 . A system according to  claim 1  wherein the loading module includes an acyl carrier protein.  
     
     
         7 . A system according to  claim 1  wherein at least the Ksq domain of said loading module corresponds to the loading module of the PKS multienzyme of oleandomycin, spiramycin, niddamycin, methmycin or monensin.  
     
     
         8 . A PKS multienzyme as expressible by the DNA of the system of  claim 1  or a variant having the ability to synthesize a said polyketide compound.  
     
     
         9 . Nucleic acid encoding the PKS multienzyme of  claim 8 .  
     
     
         10 . A vector containing nucleic acid as defined in  claim 9 .  
     
     
         11 . A transformant organism comprising a system according to  claim 1 .  
     
     
         12 . A process for producing a polyketide which comprises culturing an organism according to  claim 11  and recovering the polyketide.  
     
     
         13 . A system, multienzyme, nucleic acid, vector, organism or process according to any preceding claim wherein said polyketide is selected from 
 (a) 12- and 16-membered macrolides with acetate starter units    (b) 12, 14 and 16-membered macrolides with propionate starter units    (c) variants of avermectin, rapamycin, immunomycin and FK506 with acetate starter units or propionate starter units    (d) a polyketide wherein the starter unit gave rise to a sidechain selected from allyl and hydroxymethyl.    
     
     
         14 . A variant of a parent polyketide which differs from the parent polyketide in the side chain provided by the starter unit.  
     
     
         15 . A process for preparing a type II polyketide comprising culturing an organism containing a type II polyketide synthase (“PKS”) wherein the wild type synthase includes a CLF domain which tends to effect decarboxylation to produce an undesired starter; wherein said organism contains a PKS which has been genetically engineered to suppress the decarboxylating activity of said CLF domain.  
     
     
         16 . A method for the synthesis of polyketides which have incorporated a desired starter unit, said method comprising: 
 A) constructing in an organism a hybrid type I polyketide synthase (PKS) comprising a loading module which comprises an ATq domain and a KSq domain and at least one extension module, wherein said ATq domain is an acyltransferase domain which loads an optionally substituted malonyl-CoA residue, wherein said KSq domain is a ketosynthase domain which effects decarboxylation of the enzyme-bound optionally substituted malonate unit, and wherein at least one of said extension modules is not naturally associated with a loading module that effects decarboxylation of an optionally substituted malonyl residue; and    B) culturing said organism under conditions suitable for the production of said polyketide.    
     
     
         17 . The method according to  claim 16 , wherein said ATq is specific for a methylmalonyl-CoA residue.  
     
     
         18 . The method according to  claim 16 , wherein said ATq is specific for a malonyl-CoA residue.  
     
     
         19 . The method according to  claim 16 , wherein said ATq domain is selected from the group consisting of: 
 a) the AT of extension module 4 of the FK506 PKS gene cluster;    b) the AT of extension module 6 of the niddamycin PKS gene cluster;    c) the AT of extension module 5 of the spiramycin PKS gene cluster; and    d) the AT of extension module 5 of the monensin PKS gene cluster.    
     
     
         20 . The method according to  claim 16 , wherein said KSq domain is a natural KSq domain.  
     
     
         21 . The method according to  claim 16 , wherein said KSq domain is a KS domain from an extension module wherein the active site cysteine has been replaced with a glutamine.  
     
     
         22 . The method according to  claim 21 , wherein said KSq domain is produced via site-directed mutagenesis.  
     
     
         23 . The method according to  claim 16 , wherein said loading module further comprises an ACP domain.  
     
     
         24 . The method according to  claim 16 , wherein said hybrid type I PKS further comprises a thioesterase domain.  
     
     
         25 . The method according to  claim 16 , wherein the extension module adjacent to the loading module is not naturally associated with a loading module that effects decarboxylation of an optionally substituted malonyl residue.