Development of cloning vectors and transformation methods for Amycolatopsis

Abstract

The genus Amycolatopsis is of industrial importance, as its species are known to produce commercial antibiotics. It belongs to the family Pseudonocardiaceae and has an eventful taxonomic history. Initially strains were identified as Streptomyces, then later as Nocardia. However, based on biochemical, morphological and molecular features, the genus Amycolatopsis, containing seventeen species, was created. The development of molecular genetic techniques for this group has been slow. The scarcity of molecular genetic tools including stable plasmids, antibiotic resistance markers, transposons, reporter genes, cloning vectors, and high efficiency transformation protocols has made progress slow, but efforts in the past decade have led to the development of cloning vectors and transformation methods for these organisms. Some of the cloning vectors have broad host range (pRL series) whereas others have limited host range (pMEA300 and pMEA100). The cloning vector pMEA300 has been completely sequenced, while only the minimal replicon (pA- rep) has been sequenced from pRL plasmids. Direct transformation of mycelia and electroporation are the most widely applicable methods for transforming species of Amycolatopsis. Conjugational transfer from Escherichia coli has been reported only in the species A. japonicum, and gene disruption and replacements using homologous recombination are now possible in some strains.

Development of an Improved System for the Generation of Knockout Mutants of Amycolatopsis sp. Strain ATCC 39116

“…Previous studies done with Amycolatopsis sp. ATCC 39116 and with members of the genus Amycolatopsis in general have shown that it is quite difficult to generate deletion mutants (5,6,38). Therefore, our first goal was to improve the homologous integration of the generated suicide plasmids.…”

Identification of the chelocardin biosynthetic gene cluster from Amycolatopsis sulphurea: a platform for producing novel tetracycline antibiotics

“…The Amycolatopsis strains have often proven difficult to transform and genetically manipulate (Malhotra & Lal, 2007;Dhingra et al, 2003). We have invested very significant efforts to develop a transformation procedure for A. sulphurea, where we tested various growth media, numerous actinomycete plasmids (replicative and integrative) in combination with different procedures, such as protoplast transformation and E. coli-A.…”

Synthetic Biology in Action: Developing a Drug Against MDR-TB

“…However, with the availability of rifamycin polyketide synthase gene cluster (rifPKS) in 1998 [12] (Fig. 3) and with the development of cloning vectors and transformation system for the rifamycin producer strain A. mediterranei by our group here at the University of Delhi [13][14][15][16][17][18], the possibilities of manipulating the rifPKS to produce more effective analogues of rifamycin B were raised [16].…”

“…The prerequisites for the generation of rifamycin analogues involved developing a cloning vector and transformation system to negotiate with the bacterium A. mediterranei that produces Rifamycin B. Thus, a work initiated in 1988 resulted in crossing the first hurdle by developing a system of cloning vectors and transformation system by our team [13,15,18].…”