title: A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor.
abstract: Gene clusters found in bacterial species classified as Streptomyces encode the majority of known antibiotics as well as many pharmaceutically active compounds. A site-specific recombination system similar to those that mediate plasmid conjugation was engineered to catalyze tandem amplification of one of these gene clusters in a heterologous Streptomyces species. Three genetic elements were known to be required for DNA amplification in S. kanamyceticus: the oriT-like recombination sites RsA and RsB, and ZouA, a site-specific relaxase similar to TraA proteins that catalyze plasmid transfer. We inserted RsA and RsB sequences into the S. coelicolor genome flanking a cluster of 22 genes (act) responsible for biosynthesis of the polyketide antibiotic actinorhodin. Recombination between RsA and RsB generated zouA-dependent DNA amplification resulting in 4-12 tandem copies of the act gene cluster averaging nine repeats per genome. This resulted in a 20-fold increase in actinorhodin production compared with the parental strain. To determine whether the recombination event required taxon-specific genetic effectors or generalized bacterial recombination (recA), it was also analyzed in the heterologous host Escherichia coli. zouA was expressed under the control of an inducible promoter in wild-type and recA mutant strains. A plasmid was constructed with recombination sites RsA and RsB bordering a drug resistance marker. Induction of zouA expression generated hybrid RsB/RsA sites, evidence of site-specific recombination that occurred independently of recA. ZouA-mediated DNA amplification promises to be a valuable tool for increasing the activities of commercially important biosynthetic, degradative, and photosynthetic pathways in a wide variety of organisms.
authors: Department of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3.
authors: Takeshi
authors: T
authors: Murakami
authors: Jan
authors: J
authors: Burian
authors: Koji
authors: K
authors: Yanai
authors: Mervyn J
authors: MJ
authors: Bibb
authors: Charles J
authors: CJ
authors: Thompson
mesh_terms: D000880:Anthraquinones / Q000378:metabolism; D000900:Anti-Bacterial Agents / Q000096:biosynthesis*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015139:Blotting, Southern; D004269:DNA, Bacterial / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005784:Gene Amplification; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D005810:Multigene Family; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D013302:Streptomyces / Q000235:genetics / Q000378:metabolism; D048372:Streptomyces coelicolor / Q000235:genetics* / Q000378:metabolism; D000095622:Benzoisochromanequinones
chemical_list: D000880:Anthraquinones; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D004269:DNA, Bacterial; C013390:actinorhodin; D000095622:Benzoisochromanequinones