Formation of Streptomyces Protoplasts during Cultivation in Liquid Media with Lytic Enzyme

Many streptomycetes strains are hardly or not at all transformable via protoplasts, or there is a problem with the regeneration of protoplasts. We found that protoplasts are formed directly in cultivation media under submerged conditions in the presence of lytic enzyme. Actinophage μ1/6 endolysin and lysozyme were used in this study. Streptomyces strains were cultivated in several media with glycine and lytic enzyme for 24 and 48h. The highest amounts of protoplasts (about 3 x 10 cfu/ml of cultivation medium) together with the highest regeneration (95%) and transformation frequency (about 2 x 10 – 10 cfu/μg DNA) were obtained reproducibly in YEME medium with high sucrose content. S. aureofaciens B96, as hardly transformable strain because of difficulties with protoplast preparation and their further regeneration, was used in this study. The same procedure was applied to S. lividans 66 TK24 and S. coelicolor A3(2), streptomycetes model strains, to confirm the general use of this method. Moreover, such cultivation process was appropriate for additional quick isolation of either chromosomal as well as plasmid DNA that could be further used in recombinant DNA techniques.