title: 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.
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.
authors.affiliation: National Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, China.
authors.forename: Shan
authors.initials: S
authors.lastname: Wang
authors.forename: Ruifang
authors.initials: R
authors.lastname: Zhao
authors.forename: Kai
authors.initials: K
authors.lastname: Liu
authors.forename: Mengyi
authors.initials: M
authors.lastname: Zhu
authors.forename: Aiying
authors.initials: A
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: He
mesh_terms: D000970:Antineoplastic Agents / Q000378:metabolism*; D053898:Biosynthetic Pathways / Q000235:genetics*; D003227:Conjugation, Genetic; D017353:Gene Deletion; D018628:Gene Dosage; D020869:Gene Expression Profiling; D018014:Gene Transfer Techniques; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D036341:Intercellular Signaling Peptides and Proteins; D060847:Metabolic Engineering / Q000379:methods; D009281:Naphthalenes / Q000378:metabolism; D010455:Peptides / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D013302:Streptomyces / Q000235:genetics* / Q000378:metabolism*; D014169:Transformation, Bacterial
chemical_list: D000970:Antineoplastic Agents; D036341:Intercellular Signaling Peptides and Proteins; D009281:Naphthalenes; D010455:Peptides; C051055:azinomycin B