DNA encoding methymycin and pikromycin

Abstract

A novel pathway for the synthesis of polyhydroxyalkanoates is provided. A method of synthesizing a recombinant polyhydroxyalkanoate monomer synthase is also provided. These recombinant polyhydroxyalkanoate synthases are derived from multifunctional fatty acid synthases or polyketide synthases and generate hydroxyacyl acids capable of polymerization by a polyhydroxyalkanoate synthase. Also provided is a biosynthetic gene cluster for methymycin and pikomycin as well as a biosynthetic gene cluster for desosamine.

What is claimed is:  
     
         1 . An isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a desosamine biosynthetic gene cluster, a fragment or a biologically active variant thereof, wherein the nucleic acid sequence is not derived from the eryC gene cluster of  Saccharopolyspora erythraea  or  Streptomyces antibioticus.    
     
     
         2 . The isolated and purified nucleic acid segment of  claim 1  comprising SEQ ID NO:3.  
     
     
         3 . The isolated and purified nucleic acid segment of  claim 1  which encodes DesI, DesII, DesIII, DesIV, DesV, DesVI, DesVII, DesVIII or DesR.  
     
     
         4 . The isolated and purified nucleic acid segment of  claim 1  which is from  Streptomyces venezuelae.    
     
     
         5 . An expression cassette comprising the nucleic acid segment of  claim 1  operably linked to a promoter functional in a host cell.  
     
     
         6 . A recombinant bacterial host cell in which at least a portion of a nucleic acid sequence encoding desosamine is disrupted so as to result in a decrease or lack of desosamine synthesis, wherein the nucleic acid sequence which is disrupted is not derived from the eryC gene cluster of  Saccharopolyspora erythraea.    
     
     
         7 . The host cell of  claim 6  wherein the nucleic acid sequence which is disrupted encodes DesI, DesII, DesIII, DesIV, DesV, DesVI, DesVII, DesVIII or DesR.  
     
     
         8 . A host cell, the genome of which is augmented with the expression cassette of  claim 5 .  
     
     
         9 . A product produced by the host cell of  claim 6  which is not produced by the corresponding non-recombinant host cell.  
     
     
         10 . The product of  claim 9  which comprises a macrolide.  
     
     
         11 . An isolated and purified nucleic acid segment comprising a nucleic acid sequence comprising a macrolide biosynthetic gene cluster encoding methymycin, pikomycin, neomethymycin, narbomycin, or a combination thereof, or a biologically active variant or fragment thereof.  
     
     
         12 . The isolated and purified nucleic acid segment of  claim 11  comprising SEQ ID NO:5.  
     
     
         13 . The isolated and purified nucleic acid segment of  claim 11  comprising a biologically active variant or fragment of SEQ ID NO:5.  
     
     
         14 . The isolated and purified nucleic acid segment of  claim 11  which encodes PikR1, PikR2, PikAI, PikAII, PikAIII, PikAIV, PikAV, PikC or PikD.  
     
     
         15 . The isolated and purified nucleic acid segment of  claim 11  which is from  Streptomyces venezuelae.    
     
     
         16 . A host cell, the genome of which is augmented with the nucleic acid segment of  claim 11 .  
     
     
         17 . An isolated and purified nucleic acid sequence comprising SEQ ID NO:3, SEQ ID NO:5, a fragment thereof, the complement thereto, or which hybridizes thereto.  
     
     
         18 . An isolated polypeptide encoded by the nucleic acid segment of  claim 1  or  11 .  
     
     
         19 . A recombinant host cell in which a pikAI gene, a pikAII gene, a pikAIII gene, a pikAIV gene, a pikB gene cluster, a pikAV gene cluster, a pikC gene, a pikR1 gene, apikR2 gene, or a combination thereof, is disrupted so as to reduce or eliminate production of methymycin, neomethymycin, pikromycin, narbomycin, or a combination thereof.  
     
     
         20 . A macrolide or polyketide produced by the host cell of  claim 19  which is not produced by the corresponding non-recombinant host cell.  
     
     
         21 . An isolated and purified DNA molecule comprising a first DNA segment encoding a first module and a second DNA segment encoding a second module, wherein the DNA segments together encode a recombinant polyhydroxyalkanoate monomer synthase, and wherein at least one DNA segment is derived from the pikA gene cluster of  Streptomyces venezuelae.    
     
     
         22 . A method of providing a polyhydroxyalkanoate monomer, comprising: 
 (a) introducing into a host cell a DNA molecule comprising a DNA segment encoding a recombinant polyhydroxyalkanoate monomer synthase operably linked to a promoter functional in the host cell, wherein the recombinant polyhydroxyalkanoate monomer synthase comprises a first module and a second module, and wherein at least one DNA segment is derived from the pikA gene cluster of  Streptomyces venezuelae ; and    (b) expressing the DNA encoding the recombinant polyhydroxyalkanoate monomer synthase in the host cell so as to generate a polyhydroxyalkanoate monomer.    
     
     
         23 . A recombinant vector comprising one or more modules of a polyketide synthase wherein at least one module is from  Streptomyces venezuelae.    
     
     
         24 . The method of  claim 22  wherein the first module encodes a fatty acid synthase.  
     
     
         25 . A method of providing a polyhydroxyalkanoate monomer, comprising: 
 (a) introducing into a host cell a DNA molecule encoding a fusion polypeptide, wherein the DNA molecule comprises a first DNA segment operably linked to a promoter functional in the host cell and a second DNA segment, wherein at least one DNA segment is derived from the pikA gene cluster of Streptomyces venezuelae; and    (b) expressing the DNA in the host cell so as to generate the fusion polypeptide.    
     
     
         26 . The host cell of  claim 16  the native genome of which does not comprise an intact macrolide biosynthetic gene cluster encoding methymycin, pikomycin, neomethymycin, or narbomycin.