A system for the targeted amplification of bacterial gene clusters multiplies antibiotic yield in Streptomyces coelicolor Takeshi Murakamia,1, Jan Buriana, Koji Yanaib, Mervyn J. Bibbc, and Charles J. Thompsona aDepartment of Microbiology and Immunology, University of British Columbia, Vancouver, BC, Canada, V6T 1Z3; bBioscience Laboratories, Meiji Seika Pharma, Odawara-shi, Kanagawa 250-0852, Japan; and cDepartment of Molecular Microbiology, John Innes Centre, Colney, Norwich NR4 7UH, United Kingdom Edited* by Arnold L. Demain, Drew University, Madison, NJ, and approved August 12, 2011 (received for review May 25, 2011) Gene clusters found in bacterial species classified as Streptomyces ceutical and agricultural activities. However, when such gene encode the majority of known antibiotics as well as many phar- clusters are transferred to heterologous hosts, levels of pro- maceutically active compounds. A site-specific recombination sys- duction are often low (13–16), presumably reflecting important tem similar to those that mediate plasmid conjugation was metabolic and/or regulatory interactions with their native hosts. engineered to catalyze tandem amplification of one of these gene An important factor in the commercial production of anti- clusters in a heterologous Streptomyces species. Three genetic biotics and other pharmaceutically active secondary metabolites elements were known to be required for DNA amplification in is the cost of strain improvement for increased yields. Yield S. kanamyceticus: the oriT-like recombination sites RsA and RsB, improvement typically involves years of repeated cycles of mu- and ZouA, a site-specific relaxase similar to TraA proteins that tagenesis and screening, potentially generating mutants in both catalyze plasmid transfer. We inserted RsA and RsB sequences primary and secondary metabolism (17). For example, these into the S. coelicolor genome flanking a cluster of 22 genes (act) mutations may change primary metabolic flux to increase pre- responsible for biosynthesis of the polyketide antibiotic actino- cursor availability (17). Rate-limiting steps for antibiotic pro- rhodin. Recombination between RsA and RsB generated zouA- duction are also often associated with the secondary metabolic dependent DNA amplification resulting in 4–12 tandem copies of pathway itself, and some overproducing strains of Penicillium MICROBIOLOGY the act gene cluster averaging nine repeats per genome. This chrysogenum, S. lincolnensis, and S. kanamyceticus obtained in resulted in a 20-fold increase in actinorhodin production compared traditional screening programs contain amplifications of their with the parental strain. To determine whether the recombination antibiotic biosynthetic gene clusters (18–21). Detailed analysis of event required taxon-specific genetic effectors or generalized bac- penicillin production strains derived by repeated rounds of mu- terial recombination (recA), it was also analyzed in the heterolo- tagenesis and screening at different pharmaceutical companies gous host Escherichia coli. zouA was expressed under the control revealed that independent lineages contained a progressively of an inducible promoter in wild-type and recA mutant strains. A increasing number of tandem amplifications of the 57-kb bio- plasmid was constructed with recombination sites RsA and RsB synthetic gene cluster (18, 19). Our studies of a kanamycin-over- zouA bordering a drug resistance marker. Induction of expression producing strain of S. kanamyceticus revealed that its genome fi generated hybrid RsB/RsA sites, evidence of site-speci c recombi- contained 36 tandem copies of a 145-kb DNA sequence that recA nation that occurred independently of . ZouA-mediated DNA included the kanamycin biosynthetic gene cluster (21). A major fi ampli cation promises to be a valuable tool for increasing the group of secondary metabolites, the polyketides, have commercial activities of commercially important biosynthetic, degradative, and importance or potential applications as antibacterials, antifungals, photosynthetic pathways in a wide variety of organisms. antiparasitics, animal growth promotants, or immunosuppressants (22). Polyketide biosynthetic pathways are encoded by clusters gene duplication | mutagenesis of genes ranging in size from around 20 kb to more than 100 kb (23). These observations suggest that controlled, stable amplifi- andem amplifications of genomic DNA occur in all domains cation of entire antibiotic biosynthetic gene clusters would be Tof life including humans, plants, insects, yeast, and bacteria a valuable and generally applicable tool for engineering high- (1–4) and are proposed to be “the principle source of new genes” yielding production strains. (for references, see ref. 4). In bacteria, DNA amplification plays In Escherichia coli and Salmonella, gene duplication–amplifi- a role in antibiotic resistance, chromosome instability, gene cation (GDA) is typically initiated via recA-independent re- evolution, and increasing the level of gene expression (4–8). combination between microhomologous sequences (imperfect Regulated amplification of genes or gene clusters could also be matches of less than 20 bp) to generate a tandem duplication (3, a means of activating gene expression that is inherited over 6, 7, 24–27). Subsequent amplification requires recA-dependent subsequent generations (6). As a biotechnological tool, the in- recombination. Such amplifications are generally restricted in ducible amplification of specific regions of microbial genomes size and unstable without continuous selection or recA inacti- (amplifiable or amplified units of DNA; AUDs) could have im- vation (7, 25, 28). Although gene amplifications in Streptomyces portant applications in strain improvement for a wide variety of species have been described (29–31), the specific enzymes and complex multigene processes, such as the biosynthesis of phar- maceutically active metabolites and vitamins, bioconversions, photosynthesis, and the degradation of toxic compounds. Author contributions: T.M. and C.J.T. designed research; T.M. performed research; T.M., J.B., About half of all agriculturally and pharmaceutically impor- K.Y., M.J.B., and C.J.T. analyzed data; and T.M., J.B., K.Y., M.J.B., and C.J.T. wrote the paper. tant compounds, including the majority of antibiotics, are pro- The authors declare no conflict of interest. duced by Actinomycetes (most belonging to the Streptomyces *This Direct Submission article had a prearranged editor. “ ” genus) or fungi (9) Almost all of these secondary metabolites Data deposition: The sequence reported in this paper has been deposited in the GenBank are encoded by gene clusters (9–12). When these clusters of database (accession no. JN005928). genes are expressed, intermediates of primary metabolism are 1To whom correspondence should be addressed. E-mail: [email protected]. redirected to alternative pathways, generating antibiotics and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. other compounds with unusual structures and useful pharma- 1073/pnas.1108124108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1108124108 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 Cassette I Cassette II A 145kb zouA RsA RsB Fig. 1. Engineering zouA and its recombination kan cluster S. kanamyceticus sites RsA and RsB for amplification of an antibiotic 35kb biosynthetic gene cluster in a heterologous host. zouA RsA RsB (A) Regions of the S. kanamyceticus genome containing the three genetic elements essential for amplification (zouA, RsA, and RsB) were act cluster S. coelicolor cloned into two cassettes and targeted to flank (MT617) the actinorhodin biosynthetic gene cluster in 110kb the S. coelicolor genome by homologous re- zouA RsA RsB combination (Materials and Methods). act, acti- norhodin biosynthetic genes; kan, kanamycin biosynthetic genes. (B) A schematic representa- act cluster S. coelicolor tion of the vectors delivering cassettes I and II and (MT6h17) the genomic area that is targeted. Double-cross- over clones were identified for pAB606 and B kanamycinR pAB606 pAB1004 pAB607 by screening for viomycin resistance and loss of kanamycin resistance. Double-cross-over viomycinR zouA RsA apramycinR RsB clones for pAB1004 were confirmed by Southern blot analyses. These plasmids were used to con- Cassette I Cassette II struct strains MT17 (pAB1004), MT617 (pAB1004 and pAB606), and MT717 (pAB1004 and pAB607). Actinorhodin Biosynthesis Cluster S. coelicolor Cassette I was inserted either between positions Cassette I SCO5070-SCO5092 138,040 and 138,051 (MT617) or at position RsA 23kb 63,080 (MT6h17) (S. coelicolor genome accession number AL6458821). In both MT617 and MT6h17, viomycinR kanamycinR pAB607 cassette II was inserted at nucleotide position 167,240. sequences required to generate and maintain them are not Results known. In S. lividans, such amplifications are relatively short, Use of zouA and Flanking RsA and RsB Sites to Amplify an Antibiotic relying on two 4.7-kb repeats that amplify a 1-kb section of in- Biosynthetic Gene Cluster in S. coelicolor. To introduce zouA and tervening sequence (29, 31). RsA and RsB at sites flanking the actinorhodin biosynthetic gene A previous report described DNA amplification in S. kana- cluster (act)ofS. coelicolor, the three genetic elements were first myceticus that required zouA, two recombination sites (32), and subcloned as two cassettes (I and II) into an E. coli vector able perhaps other host-specific factors, whereas possible