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

Abstract

To select suitable genetic markers for optimizing electroporation efficiency in Amycolatopsis mediterranei, thiostrepton (tsr), erythromycin (ermE) and apramycin (am) resistance genes were used. Although tsr could not be suitably expressed in A. mediterranei, the cloning of ermE in pRL1 or its derivative (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 spontaneous mutants were observed among transformants. Under optimum conditions, maximum electroporation efficiency of 1.2 x 10(4) transformants/micrograms DNA was achieved for A. mediterranei DSM 40,773. These plasmids could also be effectively 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 strains of Amycolatopsis, we have overcome the problem of the choice of suitable selectable markers for A. mediterranei and related species.