In vivo production of cephalosporins

Abstract

The invention provides improved in vivo production of acylated cephalosporins with higher yield using expandase which is predominantly localised in the cytosol of the host cell.

1 . A method for expanding the 5-membered ring of a beta-lactam compound to form a six-membered cephem compound, comprising the steps of growing a host capable of producing said beta-lactam compound under conditions conducive thereto and allowing said host to express a heterologous expandase capable of expanding the 5-membered ring of said beta-lactam compound from a DNA fragment encoding it, so as to form the said 6-membered cephem compound, characterised in that said expandase is expressed from a DNA fragment which codes for an expandase that is localised predominantly in the cytosol of the host cell.  
   
   
       2 . The method of  claim 1 , wherein said expandase is predominantly localised in the cytosol due to a modification in the DNA fragment encoding it, whereby a microbody targeting signal has been modified.  
   
   
       3 . The method of  claim 2 , wherein the modification is of a DNA encoding an expandase endogenous to  Streptomyces clavuligerus.    
   
   
       4 . The method of  claim 1 , wherein the host is  Penicillium chrysogenum.    
   
   
       5 . The method of clam  1  wherein said expandase is altered from an unaltered form of said expandase which is that of  Streptomyces clavuligerus,  wherein said altered expandase, when expressed in a  Penicillium chrysogenum  host is localized in the cytosol of said host, wherein the C-terminal sequence of said expandase is X 1 -X 2 -X 3 , wherein X 1  is ser, cys or ala; 
 X 2  is any amino acid; and    X 3  is a non-hydrophobic amino acid.    
   
   
       6 . The method of  claim 5  where in said altered expandase X 1  is ser, X 2  is lys; and X 3  is asp.  
   
   
       7 . The method of  claim 5  where in said altered expandase X 1  is ser; X 2  is ser; and X 3  is asp.  
   
   
       8 . The method of  claim 1 , wherein the beta-lactam compound is selected from the group consisting of phenylacetyl-6-APA, phenoxyacetyl-6-APA, alpha-aminoadipoyl-6-APA, adipoyl-6-APA, glutaryl-6-APA, suberyl-6-APA, pimelyl-6-APA, or trans-b-hydromuconyl-6-APA.  
   
   
       9 . A method for making a cephem compound comprising the steps of expanding the penam ring of said beta-lactam compound in a process according to  claim 1  and recovering the cephem compound so formed.