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Coloning and expression of spiramycin polyketide synthase genes and resistance genes from S. spiramyceticus U-1941
1Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing.
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.
Insights
Researchers cloned spiramycin biosynthesis genes into a plasmid, enabling polyketide synthase deficient strains to produce antibiotics. This work enhances understanding of antibiotic gene transfer and production in Streptomyces species.
Area of Science:
- Molecular Biology
- Microbiology
- Biotechnology
Background:
- Streptomyces species are prolific producers of secondary metabolites, including antibiotics like spiramycin.
- Understanding the genetic basis of antibiotic biosynthesis is crucial for strain improvement and novel drug discovery.
Purpose of the Study:
- To isolate and characterize the genes responsible for spiramycin biosynthesis.
- To investigate the functional expression of these genes in heterologous Streptomyces hosts.
- To explore the potential for enhancing antibiotic production through genetic manipulation.
Main Methods:
- Isolation of the plasmid pCN3H8 containing spiramycin polyketide synthase genes from S. spiramyceticus.
- Restriction analysis and Southern hybridization to map gene locations.
- Subcloning into E. coli-Streptomyces shuttle vector pWHM3 to create recombinant plasmid pCG4.
- Transformation of various Streptomyces mutants and strains with pCN3H8, pCG4, or pSG3 DNA.
- Analysis of antibiotic production and resistance using UV analysis, paper chromatography, and fermentation.
Main Results:
- The plasmid pCN3H8 (44kb) contains genes homologous to actI and actIII.
- Transformation into polyketide synthase deficient mutants led to the production of midecamycin A and novel antibacterial compounds.
- Expression of spiramycin resistance was observed, and a smaller plasmid pSG3 (7.0kb) carrying the resistance gene was identified.
- Transformation with pCG4 or pSG3 DNA increased spiramycin production in S. ambofaciens.
Conclusions:
- The cloned spiramycin biosynthesis genes are functional in heterologous hosts.
- Genetic manipulation of Streptomyces strains using these plasmids can lead to the production of new or enhanced antibiotics.
- This study provides a foundation for metabolic engineering of antibiotic production in Streptomyces.