Coloning and expression of spiramycin polyketide synthase genes and resistance genes from S. spiramyceticus U-1941

Abstract

The plasmid containing the spiramycin polyketide synthase genes, pCN3H8, was obtained from the genomic library of spiramycin producing strain, S. spiramyceticus U-1941, using homologous DNA to actI and actIII genes as hybridization probes. Restriction analysis of the plasmid pCN3H8 showed that the molecular weight was 44kb. The regions homologous to the actI & actIII genes were localized by Southern hybridization, and corresponding DNA fragments were subcloned onto E. coli-Streptomyces shuttle vector pWHM3. A recombinant plasmid pCG4 was obtained. Transformation of the pCN3H8 DNA into the polyketide synthase deficient mutant of midecamycin producing strain, S. mycarofaciens subsp. No. 68, resulted in the production of midecamycin A by UV analysis. Transformation of the pCN3H8 DNA into the polyketide synthase deficient mutant of actinorhodin producing strain, S. coelicolor TK17, resulted in the production of an antibacterial compound which was neither similar to actinorhodin in color nor to spiramycin by paper chromatographic analysis. Transformant of S. lividans with pCN3H8 DNA produced an antibacterial compound as well. Resistance to spiramycin was expressed in transformants of spiramycin sensitive strain, S. griseofuscus, with pCN3H8 DNA. A plasmid pSG3 DNA with molecular weight of 7.0kb, isolated from the transformant, might be a result of in vivo deletion of pCN3H8 in S. griseofuscus. Retransformation of pSG3 DNA into S. griseofuscus confirmed that the gene of resistance to spiramycin was on the plasmid pSG3 DNA. Transformation of spiramycin producing strain, S. ambofaciens, with pCG4 or pSG3 DNA increased spiramycin production in fermentation broth.