Development of an improved cloning vector and transformation system in Amycolatopsis mediterranei (Nocardia mediterranei).

A versatile plasmid cloning vector pRL60 carrying kanamycin/neomycin resistance (km/neo), erythromycin resistance (ermE) and alpha-amylase (alpha-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, alpha-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 alpha-amy. ermE is expressed in A. mediterranei thus allowing good selection of transformants. The alpha-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 x 10(4) transformants/microg 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 alpha-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.