Efficient transformation of Micromonospora purpurea with pIJ 702 plasmid.

Abstract

The aminoglycoside antibiotic gentamicin is produced by Micromonosporapurpurea strains1 > 2).A gene cloning system for these organisms would be useful both in the study of the organization and regulation of gentamicin biosynthesis genes and, hopefully, for the improvement of the productivity of the strains.Efficient host-vector system and transformation methods have already been developed for several Streptomyces spe-cies3~7) but in the experiments of Micromonospora strains with plasmid DNArelatively low transformation frequencies were observed8>9).In order to develop an efficient transformation method for M. purpurea we studied and optimized the parameters affecting the formation, regeneration and transformation of protoplasts. Optimization of Protoplast Formation and RegenerationIn preliminary experiments none of the transformation procedures developed for Streptomyces strains3~7) proved to be suitable for efficient transformation of Micromonospora strains with the broad host range vector pIJ70210).Our M. purpurea (MNG 00209)n) did not grow well in YEMEmedium12) that is generally used for Streptomyces protoplasting.Optimal growth was obtained in JM medium containing soluble starch 2.5 %, Tryptone 0.5 %,