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Cloning and expression of midecamycin 4"-acylase gene in spiramycin producing strains
1Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences, Beijing.
Abstract:
A recombinant plasmid p66B containing the midecamycin 4"-acylase gene was obtained by cloning this gene into plasmid vetor pIJ680 from the primary clone pCN6C5, presumably harboring the midecamycin biosynthetic gene. The expression of the midecamycin 4"-acylase gene (p66B) in spiramycin producing strains resulted mainly in the production of 4"-isovalerylspiramycin. Another positive clone pCN10F5 was discovered from the genomic library of S. mycarofacians 1748 by probing with p66B DNA BamHI-BamHI 2.3kb fragment. A BamHI-BamHI 8.0kb homologous region on pCN10F5 was determined by Southern hybridization and was subcloned into plasmids pWHM3 and pIJ680. Recombinant plasmids pWF5 and p6F5 with molecular size about 15.2kb and 13.3kb, respectively, were obtained. Transformation of spiramycin producing strains with these plasmids resulted in the production of two major components. Based on their physicochemical properties and spectral evidences, component I was identified as 4"-propionylspiramycin III, and component II as 4"-propionylspiramycin II. Southern hybridization confirmed that the BamHI-BamHI 8.0kb fragment was cloned in the spiramycin producing strain. Only pCN10F5 clone was identified from the genomic library of S. mycarofaciens 1748 when the 4"-isovaleryltransferase gene of carbomycin producing strain S. thermotolerans was used as a probe in colony hybridization. It suggests that there is a difference between the 4"-acyltransferase genes in the pCN6C5 and pCN10F5 clones.
Insights
Researchers cloned the midecamycin 4"-acylase gene, leading to 4"-isovalerylspiramycin production. Further cloning yielded new plasmids that produced 4"-propionylspiramycin variants in spiramycin-producing strains, indicating distinct acyltransferase genes.
Area of Science:
- * Molecular Biology
- * Biochemistry
- * Genetics
Background:
- * Macrolide antibiotics like spiramycin are crucial therapeutics.
- * Understanding the biosynthesis of macrolides involves identifying and characterizing key genes, such as acyltransferases.
- * Genetic engineering of antibiotic-producing strains can lead to novel derivatives with potentially improved properties.
Purpose of the Study:
- * To clone and express the midecamycin 4"-acylase gene.
- * To investigate the genetic basis for acyl group modification in spiramycin biosynthesis.
- * To generate novel spiramycin derivatives through genetic manipulation.
Main Methods:
- * Gene cloning using plasmid vectors (pIJ680, pWHM3).
- * Construction of recombinant plasmids (p66B, pWF5, p6F5).
- * Bacterial transformation of spiramycin-producing strains.
- * Southern and colony hybridization for gene confirmation.
- * Analysis of antibiotic products using physicochemical and spectral methods.
Main Results:
- * The midecamycin 4"-acylase gene (p66B) expression produced 4"-isovalerylspiramycin.
- * Subcloning of a homologous region from S. mycarofaciens 1748 yielded plasmids (pWF5, p6F5) that produced 4"-propionylspiramycin II and III.
- * Hybridization studies suggested differences between acyltransferase genes in different clones.
Conclusions:
- * The cloned genes encode acyltransferases involved in modifying the macrolide antibiotic spiramycin.
- * Genetic manipulation can successfully produce novel acyl-modified spiramycin derivatives.
- * Distinct acyltransferase genes exist in different antibiotic-producing strains, offering avenues for exploring diverse macrolide biosynthesis pathways.
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