Formation, Regeneration and Efficient Plasmid Transformation of Protoplasts of the major 2-deoxystreptamine aminoglycoside producers

Abstract

Procedures for efficient protoplasts formations, regeneration and transformation of the major 2-deoxystreptamine aminoglycoside producers were carried out as prerequisites for biocombinatorial synthesis of new antibiotics. The aminogylcoside producers used in this study were Streptomyces (S.) rimosus (paromomycin), S. ribosidificus (ribostamycin), S. fradiae (neomycin), S. kanamyceticus (kanamycin), S. tenebrarius (apramycin & tobramycin), and M. olivasterospora (fortimicin B). Both pUWL218 and pUWL201PW shuttle plasmids (thiostrepton resistance) were used for protoplast transformations. Optimum incubation time and lysozyme concentration for protoplast formation were 30 min and 1-2 mg/ml for all the selected strains except for S. fradiae where it required longer incubation time (60 min) and higher lysozyme concentration (4 mg/ml). S. fradiae was found to have the lowest rate of protoplast formation and plasmid transformation efficiencies as compared to the other bacterial strains. Optimum plasmid DNA concentration was 100 -200 ng per 200 ml protoplasts for all the selected strains whereas higher concentration (500 ng) significantly increased transformation efficiency with S. fradiae. S. kanamyceticus was resistant to thiostrepton and apramycin, the two selective markers used in this work. Therefore, studying the efficiency of protoplast transformation of S. kanamyceticus has to be conducted in future using plasmids having different resistance markers.