Transformation and transfection of anthracycline-producing streptomycetes

Abstract

Streptomyces peucetius and Streptomyces strain C5, producers of anthracycline antibiotics, were converted to protoplasts from vegetatively growing mycelia. Conditions are described for maximal protoplast formation (>99%) and for regeneration frequencies of up to 13%. Streptomycete plasmids pIJ61, pIJ702, and pIJ922, from the replicons SLP1, p1.][01, and SCP2*, respectively, were isolated from Streptomyces lividans 66 and successfully introduced into S. peucetius and Streptomyces strain C5 by polyethylene glycol-mediated protoplast transformation. Frequencies of up to 106 transformations ,ug of plasmid DNA-' were achieved by these procedures. Analyses showed that the two anthracycline-producing strains can stably harbor the plasmids without deletion of plasmid sequences or loss of the plasmids for several transfers through selective media. Fragments of DNA from S. peucetius ligated into pIJ702 and introduced into Streptomyces strain C5 were stable after several transfers through selective media. Both anthracycline producers also were sensitive to infection and transfection by actinophages KC401 and KC515, clear plaque derivatives of bacteriophage +C31. Optimal conditions were determined for the transfection of S. peucetius and Streptomyces strain C5 protoplasts with 4C31 KC401 and KC515 DNA with liposome-assisted, polyethylene glycol-mediated protoplast transfection.

Protoplast formation and regeneration in Streptomyces flavopersicus

“…Seed cultures from the first stage flask were used to inoculate the second stage liquid medium containing 2% glycine and allowed to grow to the optimal growth phase for protoplasting. While for some strains the stationary phase proved to be optimal (Lampel and Strohl 1986), other strains yielded the highest titre of protoplasts when the mycelia were at the transition of logarithmic growth to the stationary phase (Hopwood 1981). In the case of S.Jlavopersicus NRRL 2820 cultures from late logarithmic phase (24 h growth) proved to generate the highest titre of protoplasts (Fig.…”

Construction of a hybrid plasmid capable of replication in Amycolatopsis mediterranei

“…Methods for gene cloning have been developed for several species of Streptomyces (1,15,20,31). Although A. mediterranei belongs to the same order (Actinomycetales), recombinant DNA techniques were not available for this organism.…”