Selection of suitable marker genes for the development of cloning vectors and electroporation in different strains of Amycolatopsis mediterranei

To select suitable genetic markers for optimizing electroporation efficienc y in Amycolatopsis mediterranei, thiostrepton (tsr), erythromycin (ermE) an d apramycin (am) resistance genes were used. Although tsr could not be suit ably expressed in A. mediterranei, the cloning of ermE in pRL1 or its deriv ative (containing am) resulted in the development of cloning vectors pRLM20 , pRLM30 and pRL90. In contrast to tsr and km (kanamycin resistance gene), ermE and am were suitably expressed in A. mediterranei strains and no spont aneous mutants were observed among transformants. Under optimum conditions, maximum electroporation efficiency of 1.2x10(4) transformants/mu g DNA was achieved for A. mediterranei DSM 40773. These plasmids could also be effec tively transferred in other strains of A. mediterranei including F1/24 and T-195. With the cloning of ermE and am and their expression in different st rains of Amycolatopsis, we have overcome the problem of the choice of suita ble selectable markers for A. mediterranei and related species.