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The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli
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Coloning and expression of spiramycin polyketide synthase genes and resistance genes from S. spiramyceticus U-1941

L Tang1, Y G Wang, X W Zhu

  • 1Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing.

Chinese Journal of Biotechnology
|January 1, 1991
PubMed

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.

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