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Streptomyces kasugaensis produces kasugamycin, an aminoglycoside antibiotic effective against rice blast disease. A derivative strain of this species has met the standards for a DNA cloning host1) and has been listed as a particularly safe Streptomyces host since 1991 in the Japan Guidelines for Recombinant DNA Experiments together with Streptomyces coelicolor A3(2), Streptomyces griseus, Streptomyces lividans and Streptomyces parvulus . However, for genetic studies of S. kasugaensis involving gene cloning and expression, the available vectors were insufficient: pIJ7022), the most widely-used cloning vector for Streptomyces, was not maintained in the cells with adequate stability3). Derivative vectors3) constructed from pSK1 and pSK24) found in S. kasugaensis MB273 carried a single selectable marker, the thiostrepton-resistance gene (tsr) from Streptomyces azureus, but another marker was required for efficient clone recognition in addition to tsr. The melanin-synthesizing melC from Streptomyces antibioticus was examined for potential use as a chromogenic selectable marker for S. kasugaensis, because this species forms no melanoid pigments on agar media5). Gene melC is composed of a typical streptomycete promoter followed by the melC operon consisting of melC1 and melC26); melC1 is involved in the copper-transfer process that activates apotyrosinase encoded by melC27). As observed with melC-harboring pIJ702, insertion of DNA fragments into the internal single cleavage sites of melC1 (e.g., BglII, SacI, and SphI) inactivates the operon, thereby resulting in the visually-recognizable, melanin-negative phenotype of host streptomycetes such as S. lividans2). It was found, however, that transformants of S. kasugaensis with pSK212, a melC-carrying derivative of pSK2, failed to induce detectable amounts of melanin pigment on agar media. This study describes melE, a hybrid chromogenic marker, and vectors pSK216 and pSK221, both of which contain melE and tsr. Because the melC operon is under the control of the “strong” promoter of the erythromycin-resistance gene (ermE)8) from Saccharopolyspora erythraea in melE, pSK216 and pSK221 induced elevated production of melanin pigment in S. kasugaensis and S. lividans. The vector construction thus established a cloning system for S. kasugaensis.

Cloning System for Streptomyces kasugaensis Using the Hybrid Melanin-Synthesizing Gene melE as a Chromogenic Marker