DEVELOPMENT OF A PLASMID-CLONING SYSTEM FOR STREPTOMYCES-VIRIDOCHROMOGENES TU494

A plasmid-cloning system was developed for Streptomyces viridochromogenes Tu494, a producer of the tripeptide antibiotic phosphinothricyl-alanyl-alanine (PTT). Parameters affecting protoplast formation and transformability of S. viridochromogenes were investigated in detail. A procedure giving rise to transformation efficiencies of 104-105 transformants per .mu.g DNA was worked out. Several Streptomyces plasmid vectors such as pIJ350, pIJ61, pEB2, pGM4 and pSW2 were tested in S. viridochromogenes. Some of these vectors (pGM4, pEB2) showed a high copy number, whereas the copy number of other (pIJ350, pIJ61 and pSW2) was markedly lower. Under non-selective conditions some of the vectors (pIJ350, pEB2) were not stably maintained, in contrast to the vectors pGM4 and pSW2 which did not require any selection pressure for stable maintenance. Therefore, the plasmid vectors pGM4 and pSW2 derived from endogenous replicons of Streptomyces ghanaensis strains represent appropriate plasmid vehicles for cloning experiments in S. viridochromogenes.