title: In vivo manipulation of the bleomycin biosynthetic gene cluster in Streptomyces verticillus ATCC15003 revealing new insights into its biosynthetic pathway.
abstract: Bleomycin (BLM), an important clinically used antitumor compound, and its analogs are challenging to prepare by chemical synthesis. Genetic engineering of the biosynthetic pathway in the producer strain would provide an efficient and convenient method of generating new derivatives of this complex molecule in vivo. However, the BLM producing Streptomyces verticillus ATCC15003 has been refractory to all means of introducing plasmid DNA into its cells for nearly two decades. Several years after cloning and identification of the bleomycin biosynthetic gene cluster, this study demonstrates, for the first time, genetic accessibility of this pharmaceutically relevant producer strain by intergeneric Escherichia coli-Streptomyces conjugation. Gene replacement and in-frame deletion mutants were created by lambdaRED-mediated PCR targeting mutagenesis, and the secondary metabolite profile of the resultant mutants confirmed the identity of the BLM biosynthetic gene cluster and established its boundaries. Ultimately, the in-frame blmD deletion mutant strain S. verticillus SB5 resulted in the production of a bleomycin intermediate. The structure of this compound, decarbamoyl-BLM, was elucidated, and its DNA cleavage activity was compared with the parent compounds.
authors.affiliation: Division of Pharmaceutical Sciences, University of Wisconsin-Madison, Madison, WI 53705, USA.
authors.forename: Ute
authors.initials: U
authors.lastname: Galm
authors.forename: Liyan
authors.initials: L
authors.lastname: Wang
authors.forename: Evelyn
authors.initials: E
authors.lastname: Wendt-Pienkowski
authors.forename: Runying
authors.initials: R
authors.lastname: Yang
authors.forename: Wen
authors.initials: W
authors.lastname: Liu
authors.forename: Meifeng
authors.initials: M
authors.lastname: Tao
authors.forename: Jane M
authors.initials: JM
authors.lastname: Coughlin
authors.forename: Ben
authors.initials: B
authors.lastname: Shen
mesh_terms: D001761:Bleomycin / Q000096:biosynthesis*; D004247:DNA / Q000378:metabolism; D004187:Disaccharides / Q000737:chemistry; D004926:Escherichia coli / Q000378:metabolism; D017353:Gene Deletion; D005818:Genetic Engineering; D008954:Models, Biological; D008956:Models, Chemical; D008957:Models, Genetic; D005810:Multigene Family; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011995:Recombination, Genetic; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000378:metabolism*
chemical_list: D004187:Disaccharides; D001761:Bleomycin; D004247:DNA