Essential role of an unknown geneaziU3in the production of antitumor antibiotic azinomycin B verified by utilizing optimized genetic manipulation systems forStreptomyces sahachiroi

Abstract

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.

Characterization of a novel DNA glycosylase fromS. sahachiroiinvolved in the reduction and repair of azinomycin B induced DNA damage

“…The orf1 gene proved to be essential for the wild type strain. However, it became easy to eliminate orf1 when the azinomycin B biosynthetic pathway was abolished by knockout of the key biosynthetic gene aziU3 ( 20 ) (Supplementary Figure S2). Obviously, the essential nature of orf1 is closely bound to production of azinomycin B.…”

“…Strains, plasmids and primer sequences used in this study are summarized in Supplementary Data. Growth and fermentation of S. sahachiroi and isolation of azinomycin B were performed as described previously ( 20 ). Streptomyces and Escherichia coli strains were cultured and manipulated as previously reported ( 21 ).…”

Genome mining of novel rubiginones from Streptomyces sp. CB02414 and characterization of the post-PKS modification steps in rubiginone biosynthesis

“…Analysis of cluster 5 revealed eight ORFs that encode proteins with high sequence similarities (identity > 66%) to proteins from spore pigment BGCs, such as the whiE gene cluster from Streptomyces coelicolor A3(2) [ 19 ], the whiEa gene cluster from Streptomyces avermitilis [ 20 ], the cur gene cluster from Streptomyces curacoi [ 21 ], the sah gene cluster from Streptomyces sahachiroi ATCC 33158 [ 22 ], the mec gene cluster from Streptomyces bottropensis [ 23 ], the whiESa gene cluster from Streptomyces aureofaciens CCM3239 [ 24 ], and the pksA gene cluster from Streptomyces collinus DSM2012 (Accession number AF293354.1, unpublished data) (Fig. 1 A).…”