Cloning System for Streptomyces Kasugaensis Using the Hybrid Melanin-Synthesizing Gene Mele As a Chromogenic Marker

Streptomyces kasugaensis, a producer of kasugamycin, is listed as a particularly safe Streptomyces host in the Japan Guidelines for Recombinant DNA Experiments. In search of a chromogenic selectable marker usable for S. kasugaensis, melanin-synthesizing melE was constructed by placement of the operon for the melanin-synthesizing gene (melC) from Streptomyces antibioticus under the control of the promoter of the erythromycin-resistance gene (ermE) from Saccharopolyspora erythraea. Combined use of melE and the thiostrepton-resistance gene from Streptomyces azureus yielded vectors pSK216 and pSK221, derivatives of pSK2 from S. kasugaensis MB273. In S. kasugaensis and Streptomyces lividans, melE-harboring pSK216 and pSK221 induced detectable amounts of melanin pigment on agar media in marked contrast to pSK2-based pSK212 carrying melC, thereby establishing an efficient cloning system for S. kasugaensis.