Recombinant narbonolide polyketide synthase

Abstract

Recombinant DNA compounds that encode all or a portion of the narbonolide polyketide synthase are used to express recombinant polyketide synthase genes in host cells for the production of narbonolide, narbonolide derivatives, and polyketides that are useful as antibiotics and as intermediates in the synthesis of compounds with pharmaceutical value.

1 . A recombinant DNA compound that comprises a coding sequence for a domain of a narbonolide PKS.  
     
     
         2 . The recombinant DNA compound of  claim 1 , wherein said domain is selected from the group consisting of a thioesterase domain, a KS Q  domain, an AT domain, a KS domain, an ACP domain, a KR domain, a DH domain, and an ER domain.  
     
     
         3 . The recombinant DNA compound of  claim 2  that comprises the coding sequence for a loading module, thioesterase domain, and all six extender modules of the narbonolide PKS.  
     
     
         4 . The recombinant DNA compound of  claim 2  that comprises a hybrid PKS.  
     
     
         5 . The recombinant DNA compound of  claim 4  wherein said hybrid PKS comprises at least a portion of a narbonolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than narbonolide.  
     
     
         6 . The recombinant DNA compound of  claim 5  wherein said second PKS gene is a DEBS gene.  
     
     
         7 . The recombinant DNA compound of  claim 6  wherein said hybrid PKS is composed of a loading module and extender modules 1 through 6 of DEBS excluding a KR domain of extender module 6 of DEBS and an ACP of extender module 6 and a thioesterase domain of the narbonolide PKS.  
     
     
         8 . A recombinant DNA compound that comprises a coding sequence for a desosamine biosynthetic gene or a desosaminyl transferase gene or a beta-glucosidase gene of  Streptomyces venezuelae.    
     
     
         9 . A recombinant DNA compound that comprises a coding sequence for a picK hydroxylase gene of  Streptomyces venezuelae.    
     
     
         10 . The DNA compound of any of claims  1 - 9  that further comprises a promoter operably linked to said coding sequence.  
     
     
         11 . The recombinant DNA compound of  claim 10 , wherein said promoter is a promoter derived from a cell other than a  Streptomyces venezuelae  cell.  
     
     
         12 . The recombinant DNA compound of  claim 11  that is a recombinant DNA expression vector.  
     
     
         13 . The expression vector of  claim 12  that expresses a PKS in Streptomyces host cells.  
     
     
         14 . A recombinant host cell, which in its untransformed state does not produce 10-deoxymethynolide or narbonolide, that comprises a recombinant DNA expression vector of  claim 12  that encodes a narbonolide PKS and said cell produces 10-deoxymethynolide or narbonolide.  
     
     
         15 . The recombinant host cell of  claim 14  that further comprises a picB gene.  
     
     
         16 . The recombinant host cell of  claim 14  that further comprises desosamine biosynthetic genes and a gene for desosaminyl transferase and produces YC17 or narbomycin.  
     
     
         17 . The recombinant host cell of  claim 16  that further comprises a picK gene and produces methymycin, neomethymycin, or picromycin.  
     
     
         18 . The recombinant host cell of any of  claim 17  that is  Streptomyces coelicolor  or  Streptomyces lividans.    
     
     
         19 . A recombinant host cell other than a  Streptomyces venezuelae  cell that expresses the picK hydroxylase gene of  S. venezuelae.    
     
     
         20 . A recombinant host cell other than a  Streptomyces venezuelae  host cell that expresses a desosamine biosynthetic gene or desosaminyl transferase gene of  S. venezuelae.    
     
     
         21 . A method for increasing the yield of a desosaminylated polyketide in a cell, which method comprises transforming the cell with a recombinant expression vector that encodes a functional beta-glucosidase gene.  
     
     
         22 . A hybrid PKS which comprises at least one domain of a narbonolide PKS.  
     
     
         23 . The hybrid PKS of  claim 22  wherein said hybrid PKS comprises at least a portion of a narbonolide PKS gene and at least a portion of a second PKS gene for a macrolide aglycone other than narbonolide.  
     
     
         24 . The hybrid PKS of  claim 23  wherein said second PKS gene is a DEBS gene.  
     
     
         25 . The hybrid PKS of  claim 24  wherein said hybrid PKS is composed of a loading module and extender modules 1 through 6 of DEBS excluding a KR domain of extender module 6 of DEBS and an ACP of extender module 6 and a thioesterase domain of the narbonolide PKS.  
     
     
         26 . A method to produce a polyketide which comprises providing starter, extender and/or intermediate ketide units to the hybrid PKS of  claim 22 .  
     
     
         27 . A polyketide produced by the method of  claim 26.