For example, the gene aziU3 shows sequence similarity only to hyp

For example, the gene aziU3 shows sequence similarity only to hypothetical proteins of unknown functions in different bacterial species. The involvement of aziU3 in the azinomycin B biosynthesis is yet to be determined. Using our optimized isocitrate dehydrogenase signaling pathway genetic manipulation systems described above that enables easier transfer of foreign DNA into S. sahachiroi, we investigated whether this gene is essential for azinomycin B biosynthesis by in-frame

deletion. A 1.73-kb upstream region and a 1.77-kb downstream region of aziU3 were cloned into pOJ260 to yield pMSB-WS09. This plasmid was classified as a suicide plasmid because of the absence of a Streptomyces replicon and the genes for site-specific integration. After introduction into Streptomyces, the plasmid could propagate only if integrated into Dabrafenib price the chromosome via the first crossover event between either pair of homologous regions to yield conjugants/transformants. In general, introduction of suicide plasmids into wild-type streptomycete is more difficult than the site-specific integrative or autoreplicative plasmids (Kieser et al., 2000). Nevertheless, conjugal transfer of our pMSB-WS09 from E. coli to S. sahachiroi was achieved at an unexpected high efficiency (10−5 conjugants per recipient). The gene aziU3 was deleted after the second crossover event between another pair of homologous regions to yield the mutant strain ΔaziU3 (Fig. 2 and Fig. S7). Bioassay

and HPLC-MS analyses demonstrated that the azinomycin B biosynthesis

was abolished when aziU3 was absent from the azi cluster (Figs 3 and 4). To rule out possible polar effects caused by gene replacement, complementation of aziU3 was performed in trans using an integrative plasmid pMSB-WS38 with aziU3 located downstream of the promoter PermE*, which is from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea. This plasmid was introduced into the deletion mutant ΔaziU3 by intergeneric conjugation to yield the complementation strain ΔaziU3::aziU3 (Fig. 2 and Fig. S7). Production of azinomycin B in the complementation strain was not only restored but also increased 24% compared with the wild-type strain. These results indubitably indicated that AziU3 was involved in the azinomycin B biosynthesis. In addition, it also showed that the promoter PermE* Carnitine palmitoyltransferase II from S. erythraea worked as a strong constitutive promoter in S. sahachiroi, which is not observed in every Streptomyces species. It was speculated that ΔaziU3::aziU3 produces higher amounts of azinomycin B than the wild-type strain because of increased aziU3 expression regulated by the strong promoter PermE*. To further increase the expression level of this gene, the plasmid pMSB-WS38 carrying one copy of aziU3 was introduced into wild-type S. sahachiroi by protoplast transformation, yielding WT::aziU3. As expected, production of azinomycin B increased further (Fig.

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