Essential role of an unknown gene aziU3 in the production of antitumor antibiotic azinomycin B verified by utilizing optimized genetic manipulation systems for Streptomyces sahachiroi.

Streptomyces sahachiroi ATCC 33158 produces the potent antitumor antibiotic azinomycin B, which is featured with a set of unusual functionalized moieties. However, the genetic analyses of azinomycin B biosynthetic pathway are hampered by the low efficiency of S. sahachiroi genetic manipulation. In this study, we developed two efficient DNA transfer systems for S. sahachiroi ATCC 33158 by optimizing a variety of parameters known to affect intergeneric conjugation and protoplast transformation. High efficiencies of 4 × 10(2) transformants per μg DNA and 2.47 × 10(-4) conjugants per recipient were achieved when using the integrative vector pJTU2554. With the use of these improved genetic manipulation systems, aziU3 was discovered to play a key role in the biosynthesis of azinomycin B. In-frame deletion and complementation experiments demonstrated clearly that aziU3 is essential for azinomycin B biosynthesis. Changing the native promoter and insertion of an additional aziU3 gene copy resulted in two mutant strains over-producing azinomycin B. Real-time PCR verified that overexpression of aziU3 significantly improved the azinomycin B production in these mutant strains.