Polyketides and Their Synthesis and Use

Abstract

The rapamycin gene cluster is an example of a gene cluster which includes a gene (rapL) leading to the formation of a precursor compound (pipecolic acid, in this case) which is required for inclusion in the larger product (rapamycin) produced by the enzymes encoded by the cluster. We have produced a mutant strain containing a rapamycin gene cluster in which the rapL gene is disabled. The strain does not naturally produce rapamycin but does so if fed with pipecolic acid. By feeding with alternative carboxylic acids we have produced variants of rapamycins. Tests have shown biological activity.

1 . A process of modifying a gene cluster involved in the biosynthesis of a polyketide, said gene cluster including a gene (“the precursor gene”) responsible for the production of an enzyme which is responsible for the production of a precursor compound which is incorporated into said polyketide; said process comprising the step of deleting or inactivating said precursor gene. 
   
   
       2 . A process according to  claim 1  wherein said process of deleting or inactivating said precursor gene employs phage-mediated gene replacement. 
   
   
       3 . A process according to  claim 1  or  claim 2  wherein the gene cluster is the gene cluster for the production of rapamycin in  S. hygroscopicus  and the precursor gene is the rapL gene whose product is responsible for the production of L-pipecolate. 
   
   
       4 . A process for producing a polyketide comprising modifying a gene cluster by the process of  claim 1 ,  2  or  3  and expressing the modified gene cluster to produce polyketide synthase enzymes which act in the presence of a variant precursor compound which is incorporated so that a variant polyketide is produced. 
   
   
       5 . A process according to  claim 4  as appendant on  claim 3  wherein the variant precursor compound is selected from L-proline, L-trans-4-hydroxyproline, L-cis-4-hydroxyproline, L-cis-3-hydroxyproline and trans-3-aza-bicyclo[3,1,0]hexane-2-carboxylic acid. 
   
   
       6 . A compound selected from prolyl-rapamycin, 4-hydroxy-propyl rapamycin, 4-hydroxyproplyl-2,6-demethoxy-rapamycin, 3-hydroxy-prolyl-rapamycin 3-hydroxy-prolyl-2,6-demethoxy-rapamycin, and trans-3-aza-bicyclo[3,1,0]hexane-2-carboxylic acid rapamycin. 
   
   
       7 . A pharmaceutical composition comprising a compound of  claim 6 . 
   
   
       8 . Use of a compound of  claim 6  in the manufacture of an immunosuppressant composition. 
   
   
       9 . A modified gene cluster as produced by the process of any of  claims 1 - 3 . 
   
   
       10 . A vector containing the gene cluster of  claim 9 . 
   
   
       11 . A microorganism containing the gene cluster of  claim 9  and capable of expressing it.