Optimum conditions for efficient transformation of Streptomyces venezuelae protoplasts

Optimum conditions for protoplast regeneration and transformation of Streptomyces venezuelae ETH14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10(7) transformants/micrograms DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30 degrees -32 degrees C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10(5) regenerating protoplasts per plate. Higher protoplasts densities considerably decreased the regeneration frequency of the transformants.