A versatile plasmid cloning vector pRL60 carrying kanamycin/neomycin resistance (km/neo), erythromycin resistance (ermE) and α-amylase (α-amy) marker genes that replicates in various Amycolatopsis mediterranei strains and Escherichia coli has been constructed. This cloning vector has been derived from a hybrid plasmid pRL50, which was developed by cloning ermE from pIJ4026 into a pRL1 derivative pULAM2. While cloning ermE into the BamHI site of pULAM2, only a hybrid plasmid pRL50 with an additional copy of pULAM2 was selected. Thus pRL50 (18.7 kb) contained two copies each of the km/neo, α-amy, and one copy of ermE. When pRL50 was transformed into A. mediterranei DSM 40773 through electroporation and selected under erythromycin resistance, the plasmid underwent a spontaneous deletion of 8.5 kb fragment resulting in the formation of plasmid pRL60. pRL60 (10.2 kb) is a shuttle vector between A. mediterranei and E. coli with three marker genes: km/neo, ermE and α-amy. ermE is expressed in A. mediterranei thus allowing good selection of transformants. The α-amy gene of pRL60 is also expressed in A. mediterranei DSM 40773 and its activity can be easily detected on starch containing medium after iodine staining. Most critical parameters evaluated for electrotransformation using pRL60 in A. mediterranei were growth phase, electrical field strength, pulse length, pretreatment of mycelia with lysozyme and use of salt free water. At optimized parameters, a transformation efficiency of 4.0×104 transformants/μg DNA was reproducibly achieved for A. mediterranei DSM 40773. pRL60 could also be transformed into A. mediterranei DSM 43304, DSM 46095, MTCC-17 and in mutants F1/24 and T-195, (derived from an industrial strain of A. mediterranei N813). The α-amy of pRL60 conferred an amylolytic phenotype to all these strains. With the development of pRL60 and a reproducible transformation protocol, the application of recombinant DNA techniques to these industrial microorganisms has now become feasible.

Rifamycins are primarily produced by Gram-positive bacterium Amycolatopsis mediterranei, which belongs to the order Actinomycetales. These antibiotics, apart from their application against pathogens of tuberculosis and leprosy, have also been found to be effective against several other pathogens including Mycobacterium avium and Pneumococcus. Because of the importance of rifamycin, the producer strain A. mediterranei has been genetically manipulated since 1957 in order to develop a strain that can either produce larger amounts of rifamycin or derivatives of rifamycin. In this article, the importance of the producer strain, traditional methods (mutations and recombination) of strain improvement, their limitations, and the development of a cloning vector and transformation methods that have made recombinant DNA techniques accessible for genetic manipulations of A. mediterranei are discussed.