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to discover natural-product gene clusters because the analysis of a A genomics-guided relatively small number of GSTs provides reasonable assurance of full genome representation. For example, analysis of 1,000 GSTs approach for discovering from a genome of 8.5 Mbp (the approximate size of the genome of an antibiotic-producing actinomycete) provides DNA sequence sampling every 8.5 kbp (assuming random library coverage). and expressing cryptic Given that natural-product gene clusters range in size from 20 to 200 kbp6,7 (C.F., unpublished data), it is expected that any given metabolic pathways gene cluster will be represented by anywhere from 2 to more than 20 of 1,000 GSTs analyzed. To date, we have used the genome scan- ning approach to successfully identify more than 450 natural- Emmanuel Zazopoulos1, Kexue Huang1, product gene clusters in a variety of actinomycetes (C.F., unpub- Alfredo Staffa1, Wen Liu2, Brian O. Bachmann1, lished data). Koichi Nonaka2, Joachim Ahlert2,3, Jon S. Thorson2,3, We used the genome scanning method to isolate 2,4 1 biosynthesis genes from a variety of actinomycete strains known Ben Shen , and Chris M. Farnet * to produce , a potent class of antitumor antibiotics8.The enediynes induce irreversible DNA damage by a mechanism that Published online 21 January 2003; doi:10.1038/nbt784 involves cycloaromatization of the warhead chromophore (Fig. 2A) to form highly reactive benzenoid diradicals that strip hydro- Genome analysis of actinomycetes has revealed the presence gen atoms from the sugar phosphate backbone of the DNA helix9. of numerous cryptic gene clusters encoding putative natural We chose the dynemicin and macromomycin biosynthesis gene products1,2. These loci remain dormant until appropriate chem- clusters to demonstrate the effectiveness of the genome scanning ical or physical signals induce their expression. Here we method. Comparison with the C-1027 (ref. 3), calicheamicin4, and demonstrate the use of a high-throughput genome scanning neocarzinostatin (W. L., K.N., L. Nie, J. Bae, and B.S., unpublished http://www.nature.com/naturebiotechnology method to detect and analyze gene clusters involved in natur- data) gene clusters reveals that the homology among all these loci al-product biosynthesis. This method was applied to uncover is limited to a set of five genes, including the gene encoding PKSE, biosynthetic pathways encoding enediyne antitumor antibiotics that form a putative “warhead gene cassette” (Fig. 2B). The con- in a variety of actinomycetes. Comparative analysis of five served genes are generally arranged in a presumed operon with biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS)3,4. The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring struc- ture (or “warhead”) found in all enediynes3,4. Genome scanning analysis indicates that the enediyne warhead cassette is wide- ly dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, sug- gesting that the range of enediyne natural products may be © Group 2003 Nature Publishing much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.

We have developed a high-throughput genome scanning method to discover metabolic loci independently of their expression. This approach takes advantage of the fact that the genes required for secondary metabolite biosynthesis are typically clustered together in a bacterial genome5. A shotgun DNA sequencing approach is used to generate short (700 bp) random genome sequence tags Figure 1. A diagrammatic view of the genome scanning method for (GSTs) from a library of genomic DNA prepared from a microor- high-throughput discovery of natural-product biosynthetic gene clusters. Natural-product biosynthetic genes (in color) are clustered in ganism. GSTs derived from genes that are likely to be involved in the bacterial genome (for simplicity, only a single gene cluster is the biosynthesis of natural products are identified by sequence shown). High-molecular-weight genomic DNA is randomly fragmented comparisons to a database of microbial gene clusters known to be and small fragments are used to prepare a genome sampling library involved in natural-product biosynthesis. Selected GSTs are then (GSL) in a plasmid vector while large fragments are used to prepare a cluster identification library (CIL) in a cosmid or BAC vector. Gene used to design screening probes to identify cloned subgenomic sequence tags (GSTs) are generated from the GSL clones using a fragments (for example, cosmids or bacterial artificial chromo- universal primer located in the plasmid vector. The GSTs are compared somes (BACs)) containing the genes of interest as well as the to a database of natural-product biosynthetic genes to identify tags neighboring genes that together may constitute a biosynthetic derived from genes involved in natural-product biosynthesis (“hot” GSTs, colored inserts; step 1). These genes are then used as probes to gene cluster (Fig. 1). Genome scanning provides an efficient way identify CIL clones containing the corresponding genes as well as their neighboring genes (“hot” CIL clones). Overlapping CIL clones may be identified by restriction fragment length mapping or during the 1Ecopia BioSciences, Inc., 7290 Frederick Banting, Montreal, Quebec H4S subsequent sequencing step. The hot CIL clones are randomly 2A1, Canada. 2Division of Pharmaceutical Sciences, 3Laboratory for fragmented and used to prepare a second plasmid library that provides Biosynthetic Chemistry, and 4Department of Chemistry, University of templates for sequencing (step 2). Sequencing and assembly of the Wisconsin, Madison, WI 53706. *Corresponding author selected CIL clones result in a complete natural-product gene cluster ([email protected]). that is then annotated and entered into the database (step 3).

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A HO D We compared the other war- O HO head cassette proteins to protein H3CO SSSCH3 HO OR R O OH 1 OH 2 H 007A O N sequences present in the O O S NH O CH I O 3 O H CH3 GenBank nonredundant data- H3C N O CH3 009C H3COOC base to assess putative functions. HOOC OCH3 O 1 2 R1, R2 = sugars 028D One protein family (TEBC) is

CH2 similar to the 4-hydroxybenzoyl- O 054A H3CO O N CoA thioesterase of Pseudomonas H CH3 O O O O 059A sp. strain CBS-3 in regions of the O O O O protein that have been shown to H C O O 3 O 132H H C HO have an important role in cataly- OH 3 O O O (H3C)2N O 11 CH3HN CH3 OH 135E sis and thus may be involved in OH NH2 HO O Cl polyketide chain release, cycliza- CH3 OH 145B tion, or both (see Supplementary 3 4 Fig. 3 online). Three families of B E unknown proteins (UNBL, DYNE 046E UNBV, and UNBU) show no sig- nificant homology to proteins in CALI 100B the public databases and there- MACR 171B fore represent novel protein fam- ilies that appear to be specific to NEOC enediyne biosynthetic loci. C-1027 Structural analysis of the UNBV PKSE TEBC UNBL UNBV UNBUUNBU proteins predicts that they are http://www.nature.com/naturebiotechnology secreted proteins with N-termi- C ACP nal signal sequences, whereas the NH2 KS AT ? KR DH ? PPTE COOH UNBU proteins are predicted to be integral membrane proteins Figure 2. Chemical structures of enediynes and genes involved in warhead formation. (A) The structures of with seven or eight putative the enediynes dynemicin (1), (2), neocarzinostatin (3), and C-1027 (4). The common membrane-spanning alpha cyclododecylpolyene skeleton found in all warheads is highlighted in red. The complete structure of helices (see Supplementary Figs. macromomycin has yet to be elucidated; however, the limited structural information available is consistent 4–6 online). Although the func- with a chromophore ring system similar to that found in C-1027 (ref. 20). (B) Organization of the warhead gene cassettes found in the dynemicin (DYNE), calicheamicin (CALI), macromomycin (MACR), tions of the TEBC, UNBL, UNBV, neocarzinostatin (NEOC), and C-1027 loci. (C) Domain organization of the warhead PKS, consisting of KS and UNBU proteins remain (ketoacyl synthase), AT (acyl transferase), ACP (acyl carrier protein), KR (keto reductase), DH unknown, their strict association ′ (dehydratase), and PPTE (4 -phosphopantetheinyl transferase). (D) Organization of the warhead cassette with the warhead PKS and their genes found in loci from actinomycete strains not previously reported to produce enediyne natural products. (E) Warhead cassette genes from actinomycete strains newly isolated from soil samples. 007A, locus found presence in all enediyne biosyn- in Amycolatopsis orientalis; 009C, locus found in Streptomyces ghanaensis; 028D, locus found in thetic loci strongly suggest that © Group 2003 Nature Publishing Kitasatosporia sp.; 054A, locus found in Micromonospora megalomicea subsp. nigra; 059A, locus found in they have essential roles in the Streptomyces cavourensis subsp. washingtonensis; 132H, locus found in Saccharothrix aerocolonigenes; formation, stabilization, or trans- 135E, locus found in Streptomyces kaniharaensis; 145B, locus found in Streptomyces citricolor. Loci 046E, 100B, and 171B were found in new actinomycete isolates (Ecopia BioSciences Inc.). port of the enediyne warhead. We used the genome scanning method to isolate natural-prod- unidirectional transcription and frequent overlap of translational uct biosynthetic loci from a variety of actinomycete strains that start and stop codons, suggesting that their gene products are have been reported to produce various classes of natural products coordinately expressed and functionally related. As these are the but not enediyne compounds. Out of 50 actinomycete strains ana- only genes common to all enediyne loci analyzed to date, we pro- lyzed, eight (16%) were found to contain biosynthetic loci con- pose that they constitute a functional unit responsible for the bio- taining the enediyne warhead cassette (Fig. 2D), indicating that genesis of the warhead, the single structural feature that is found these strains could potentially produce enediyne natural products. in all of the known enediynes9 (Fig. 2A). This finding is surprising, as none of the eight strains was previ- The PKSEs are likely to generate the carbon skeleton of the war- ously reported to produce enediynes, and it indicates that head by catalyzing iterative cycles of acyl-coenzyme A (acyl-CoA) enediyne biosynthetic loci occur at an unexpectedly high frequen- condensation, ketoreduction and dehydration, using an acyl carri- cy in the actinomycetes. Enediyne loci occurred at a similar fre- er protein (ACP) domain as a covalent attachment site for the quency in actinomycete strains newly isolated from soil samples: 3 growing carbon chain. The PKSEs contain enzymatic domains out of 20 (15%) randomly selected strains were found to harbor characteristic of known PKSs, including ketoacyl synthase (KS), biosynthetic loci containing enediyne warhead cassette genes (Fig. acyltransferase (AT), ketoreductase (KR), and dehydratase (DH) 2E). It is notable that the strong conservation of the enediyne war- domains, as well as ACP domains3,4 (Fig. 2C and Supplementary head gene sequence and gene order holds across several actino- Fig. 1 online). Additional analysis of the PKSE sequences mycete genera (Actinomadura, Amycolatopsis, Kitasatosporia, described here further revealed a previously unidentified domain Micromonospora, Saccharothrix, Streptomyces). in the C-terminal region of the protein that is similar to 4′-phos- Finally, we validated enediyne production by culturing strains phopantetheinyl transferases10 (PPTases) and is likely to be harboring warhead gene cassettes in a variety of fermentation involved in post-translational autoactivation of the PKSE (Fig. 2C media to test for activity in the biochemical induction assay (BIA), and Supplementary Figs. 1, 2 online). a modified prophage induction assay that detects agents that dam-

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A cates that additional classes of enediyne natural products remain to be discovered. The genome sequences of two actinomycetes, Streptomyces coelicolor1 and Streptomyces avermitilis2, reveal several sets of apparently cryptic natural-product gene clusters, suggesting that these well-studied strains may produce a greater number of bioac- tive compounds than has been detected by fermentation broth analysis16. We have demonstrated here that standard fermentation broth screening failed to identify many bacterial strains that can produce enediyne antibiotics. The ability of actinomycetes to pro- duce antibiotics and other bioactive natural products has appar- B ently been greatly underestimated. This work demonstrates the utility of genome analysis in discovering hidden metabolic poten- tial and directing rational approaches for the expression, detec- tion, and purification of new bioactive natural products.

Experimental protocol Genome scanning. The genomes of the dynemicin-producing organism (Micromonospora chersina strain M956-1, ATCC 53710) and the macro- momycin-producing organism (Streptomyces macromyceticus strain M480- M1, NRRL B-5335) were analyzed by genome scanning (described in patent application CA 2,352,451). Briefly, high-molecular-weight genomic DNA was prepared from each organism17 and used to generate a small- insert genomic sampling library (GSL) and a large-insert cluster identifi- cation library (CIL). Both libraries contain randomly fragmented genomic http://www.nature.com/naturebiotechnology Figure 3. Enediyne production measured by the BIA. The presence of DNA and therefore are representative of the entire genome. For the gener- β inhibition zones surrounded by blue rings is indicative of -galactosidase ation of the GSL, genomic DNA was sonicated and fragments of 1.5–3 kbp β induction due to DNA damage. At higher dilutions, only -galactosidase were prepared by agarose gel electrophoresis and cloned into plasmid vec- activity is observed, as the DNA damaging activity of the enediynes occurs at concentrations that are not bactericidal12. (A) BIA activity from tors. For the generation of the CIL, genomic DNA was fragmented to a size microorganisms producing calicheamicin, dynemicin, macromomycin, range of 30–50 kbp by partial digestion with the restriction endonuclease and neocarzinostatin and (B) from Amycolatopsis orientalis (007), Sau3A1 before being cloned into cosmid vectors. One thousand gene Streptomyces ghanaensis (009), and Streptomyces citricolor (145), sequence tags (GSTs) (average read length, 700 bp) were obtained from organisms not previously reported to produce enediynes, as well as from each GSL, translated into amino acid sequence, and compared to a propri- two new actinomycete isolates, 046 and 171. Media AA, YA, and ZA do etary database of microbial natural-product biosynthetic loci (DECIPHER not support enediyne production. Database, Ecopia BioSciences Inc., Montreal, Canada; http://www.ecopi- abio.com) using the basic local alignment search tool protein database 12 (BLASTP) software (http://www.ncbi.nlm.nih.gov/) to identify gene age DNA and is commonly used to assay enediyne production .In sequences likely to be involved in the production of natural products. The most media these strains did not have detectable BIA activity. efficiency of the genome scanning method depends in part on the ability to However, all strains produced BIA activity when grown in special- distinguish genes involved in natural-product biosynthesis from those © Group 2003 Nature Publishing ized media selected for their ability to support enediyne produc- involved in primary metabolism, and thus will vary according to the size tion (Fig. 3). These results provide strong evidence that these and breadth of the database used for comparison. The probability that a strains are able to produce enediyne natural products and that the particular gene cluster will be identified by analysis of a given number of expression of enediyne biosynthetic loci is restricted to certain fer- GSTs is improved if the database contains a large number of gene clusters mentation conditions. representing a broad range of natural-product classes. The DECIPHER Although all of the 11 loci encoding unknown enediynes con- database was initially populated with gene clusters representing a diverse tain the conserved warhead cassette genes, these loci differ consid- range of natural product classes collected from public databases such as erably from one another. They also differ from the loci that GenBank, and subsequently enriched with gene clusters discovered at Ecopia. Selected gene sequences were used to design screening probes to encode the structurally characterized enediynes in the surround- identify cosmids containing putative natural-product gene clusters from ing genes, which probably generate the structural units appended the CIL. Selected cosmids were sequenced by the shotgun method, and to the warhead chromophore (unpublished data). This may indi- overlapping cosmids were identified by using the cosmid end sequences as cate that each locus potentially encodes a different enediyne nat- probes to screen the CILs. ural product. Considering that a total of only 11 members of the Genome scanning was also used to isolate natural-product biosynthetic enediyne family have been described to date9, it is likely that some loci from 50 previously isolated actinomycete strains as well as from 20 of the unknown loci described here produce new classes of new actinomycete strains isolated from soil samples. Actinomycete strains enediynes. As the enediynes are the most potent antitumor agents used to isolate natural-product biosynthetic loci include Amycolatopsis ori- ever discovered8, the discovery of new classes holds great interest. entalis ATCC 43491 (vancomycin producer), Streptomyces ghanaensis While they are too toxic for systemic use in unmodified form, the NRRL B-12104 (moenomycin producer), Kitasatosporia sp. CECT 4991 enediynes have proven to be effective anticancer drugs when con- (taxane producer), Micromonospora megalomicea subsp. nigra NRRL 3275 jugated to polymers or antibodies. For example, a polymer-conju- (megalomicin producer), Streptomyces cavourensis subsp. washingtonensis NRRL B-8030 (chromomycin producer), Saccharothrix aerocolonigenes gated form of neocarzinostatin has been used clinically to treat 13 ATCC 39243 (rebeccamycin producer), Streptomyces kaniharaensis ATCC hepatoma in Japan since 1994 , whereas a calicheamicin–anti- 21070 (coformycin producer), Streptomyces citricolor IFO 13005 (aris- CD33 antibody conjugate (Mylotarg) was approved in the United teromycin and neplanocin A producer). Enediyne biosynthetic loci were 14 States in 2000 for the treatment of acute myelogenous leukemia . identified by the presence of the conserved enediyne warhead cassette In addition, several C-1027–antibody conjugates are currently genes as well as other genes frequently found in biosynthetic loci encoding under clinical evaluation as anticancer drugs15. This work indi- other natural-product classes (data not shown). The neocarzinostatin

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locus was cloned from Streptomyces carzinostaticus subsp. neocarzinostati- agar containing 0.7 mg/ml of X-gal was added onto the plate and color cus ATCC 15944, sequenced by the shotgun method, and confirmed to development was observed within 30 min. BIA activity was tested on seri- direct neocarzinostatin biosynthesis by gene inactivation and complemen- al dilutions of methanol extracts for microorganisms producing tation experiments (W. L., K.N., L. Nie, J. Bae, and B.S., unpublished calicheamicin and dynemicin as well as for microorganisms 046, 145, and data). 171. Serial dilutions of supernatants from microorganisms producing macromomycin and neocarzinostatin as well as from microorganisms 007 Protein homology analysis. To identify the PKSE PPTase domain, the C- and 009 were assayed for BIA activity. All production media used in this terminal regions of the PKSEs from the neocarzinostatin, calicheamicin study were assayed alone and shown to be negative for BIA activity (data and macromomycin biosynthetic loci were analyzed for their folding using not shown). secondary structure predictions and solvation potential information18. Comparison searches using a database of known three-dimensional struc- GenBank accession numbers. The DNA and protein sequences described tures of proteins revealed similarities with Sfp, the 4′-phosphopantetheinyl here are deposited in GenBank under accession numbers AF548580, transferase from the Bacillus subtilis surfactin biosynthetic locus19 (PDB id: AF548581, and AF546139–AF546157. 1QR0). Protein alignments based on secondary structure predictions as well as identification of conserved amino acids important for cofactor Note: Supplementary information is available on the Nature binding can be found in the Supplementary Figure 2 online. Amino acid Biotechnology website. sequence alignments of the PKSE, TEBC, UNBL, UNBV, and UNBU pro- teins from the calicheamicin, macromomycin, dynemicin, C-1027, and Acknowledgments neocarzinostatin biosynthetic loci can also be found in the Supplementary We thank S. Mercure, V. Dodelet, and M. Piraee for helpful discussions and Figures 1 and 3–6. Where applicable, putative functions for these proteins J. McAlpine for critical reading of the manuscript. B.S. is a recipient of a NSF were assessed by comparison to protein sequences present in the GenBank CAREER Award (MCB9733938) and a NIH Independent Scientist Award nonredundant database using the BLASTP software and by subcellular (AI51689). Enediyne studies in the Shen lab are supported in part by NIH protein localization prediction using the PSORT program available at grant CA78747. Research in the Thorson lab is supported in part by NIH http://psort.nibb.ac.jp./ grants CA84347, GM58196, and AI52218. J.S.T. is an Alfred P. Sloan Fellow. Fermentation and activity screening. Organisms were initially grown in Competing interests statement 25 ml of TSB17 seed medium for 60 h at 28 °C and then diluted 30-fold in The authors declare that they have no competing financial interests. http://www.nature.com/naturebiotechnology 25 ml production medium. Production medium for calicheamicin was Received 21 October 2002; accepted 4 December 2002 composed of 20 g of sucrose, 2 g of Bactopeptone (Becton Dickenson, Sparks, MD), 5 g of cane molasses, 0.1 g of FeSO4•7H2O, 0.2 g of 1. Bentley, S.D. et al. Complete genome sequence of the model actinomycetes MgSO4•7H2O, 0.5 g of KI, and 5 g of CaCO3 per liter. Production medium Streptomyces coelicolor A3(2). Nature 417, 141Ð147 (2002). for macromomycin was composed of 40 g of glucose, 5 g of dried yeast, 1 g 2. Omura, S. et al. Genome sequence of an industrial microorganism of K2HPO4, 1 g of MgSO4,1 g ofNaCl,2 g of(NH4)2SO4, 2 g of CaCO3,1 Streptomyces avermitilis: deducing the ability of producing secondary metabo- mg of FeSO4•7H2O, 1 mg of MnCl2•4H2O, 1 mg of ZnSO4•7H2O, and 0.5 lites. Proc. Natl. Acad. Sci. USA 98, 12215Ð12220 (2001). 3. Liu, W. Christenson, S.D., Standage, S. & Shen, B. Biosynthesis of the mg of NaI per liter. Production medium for dynemicin was composed of enediyne antitumor antibiotic C-1027. Science 297, 1170Ð1173 (2002). 10 g of corn starch, 5 g of Pharmamedia (Southern Cotton Oil Co., 4. Ahlert, J. et al. The calicheamicin gene cluster and its iterative type I enediyne Memphis, TN), 1 g of CaCO3, 0.05 g of CuSO4•5H2O, and 0.5 mg of NaI PKS. Science 297, 1173Ð1176 (2002). per liter. Production medium for neocarzinostatin was composed of 40 g 5. Martin, J.F. & Liras, P. Organization and expression of genes involved in the biosynthesis of antibiotics and other secondary metabolites. Annu. Rev. of glucose, 15 g of casamino acids, 5 g of NaCl, 2 g of CaCO3,1 g of Microbiol. 43, 173Ð206 (1989). K2HPO4 and 12.5 g of MgSO4 per liter. Selective media supporting 6. Beyer, S., Distler, J. & Piepersberg, W. The str gene cluster for the biosynthesis enediyne production in organisms not previously reported to express of 5′-hydroxystreptomycin in Streptomyces glaucescens GLA.0 (ETH 22794): new operons and evidence for pathway-specific regulation by StrR. Mol. Gen. enediyne compounds and new actinomycete isolates were as follows. For Genet. 250, 775Ð784 (1996). © Group 2003 Nature Publishing A. orientalis (007), medium was as described for calicheamicin produc- 7. Schwecke, T. et al. The biosynthetic gene cluster for the polyketide immunosup- tion. For S. ghanaensis (009), production medium was composed of 30 g pressant rapamycin. Proc. Natl. Acad. Sci. USA 92, 7839Ð7843 (1995). 8. Doyle, T.W. & Borders, D.B. Enediyne antitumor antibiotics. in Enediyne of glycerol, 15 g of distiller’s solubles, 10 g of Pharmamedia, 10 g of fish Antibiotics as Antitumor Agents (eds. Borders, D.B. & Doyle, T.W.) 1Ð15 (Marcel meal, and 6 g of CaCO3 per liter. For S. aerocolonigenes (132), S. kani- Dekker, New York, 1995). haraensis (135), and Ecopia strain 171, production media were composed 9. Smith, A.L. & Nicolaou, K.C. The enediyne antibiotics. J. Med. Chem. 39, of 60 g of molasses, 20 g of soluble starch, 20 g of fish meal, 0.1 g of 2103Ð2117 (1996). 10. Walsh, C.T., Gehring, A.M., Weinreb, P.H., Quadri, L.E. & Flugel, R.S. Post- CuSO4•5H2O, 0.5 mg of NaI, and 2 g of CaCO3 per liter. For S. citricolor translational modification of polyketide and nonribosomal peptide synthases. (145) and Ecopia strain 046, the production medium was composed of 10 Curr. Opin. Chem. Biol. 1, 309Ð315 (1997). 11. Benning, M.M. et al. The three-dimensional structure of 4-hydroxybenzoyl-CoA g of glucose, 10 g of starch, 15 g of soybean meal, 1 g of K2HPO4,3 g of thioesterase from Pseudomonas sp. Strain CBS-3. J. Biol. Chem. 273, NaCl, 1 g of MgSO4•7H2O, 7 mg of CuSO4•5H2O, 1 mg of FeSO4•7H2O, 8 33572Ð33579 (1998). mg of MnCl2•4H2O, and 2 mg of ZnSO4•5H2O per liter. For S. cavourensis 12. Elespuru, R.K. & Yarmolinsky, M.B. A colorimetric assay of lysogenic induction subsp. washingtonensis (059), production medium was composed of 20 g designed for screening potential carcinogenic and carcinostatic agents. of glucose, 5 g of Bactopeptone, 5 g of beef extract, 5 g of NaCl, 3 g of yeast Environ. Mutagen. 1, 65Ð78 (1979). 13. Maeda, H. The clinical effects of neocarzinostatin and its polymer conjugate, extract, and 2 g of CaCO3 per liter. Examples of media not supporting SMANCS. in Enediyne Antibiotics as Antitumor Agents (eds. Borders, D.B. & enediyne production include media AA (10 g of glucose, 40 g of corn dex- Doyle, T.W.) 363Ð380 (Marcel Dekker, New York, 1995). trin, 15 g of sucrose, 10 g of casein hydrolysate, 1 g of MgSO •7H O, and 2 14. Sievers, E.L. & Lineberger M. Mylotarg: antibody-targeted 4 2 comes of age. Curr. Opin. Oncol. 13, 522Ð527 (2001). g of CaCO3 per liter) and CECT media 32 and 131 (Colección Española de 15. Brukner, I. C-1027 Taiho Pharmaceutical Co. Ltd. Curr. Opinion Oncologic, Cultivos Tipo, Valencia, Spain) herein referred to as media YA and ZA, Endocrine & Met. Invest. Drugs 2, 344Ð352 (2000). respectively. 16. Chater, K.F. & Bibb, M.J. Regulation of bacterial antibiotic production. In ° Biotechnology vol. 7: Products of Secondary Metabolism (eds. Kleinkauf, H. & Production cultures (25 ml) were incubated for 7 d at 28 C under con- von Döhren, H.) 57Ð105 (VCH Press, Weinheim, Germany, 1997). stant agitation. Culture (2 ml) was removed and clarified by centrifuga- 17. Kieser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. & Hopwood, D.A. Practical tion to provide supernatant samples. The rest of the culture (supernatant Streptomyces Genetics (The John Innes Foundation, Norwich, UK, 2000). and mycelia) was extracted with an equal volume of methanol under agi- 18. Kelley, L.A., MacCallum, R.M. & Sternberg M.J. Enhanced genome annotation using structural profiles in the program 3D-PSSM. J. Mol. Biol. 299, 499Ð520 tation for 30 min. Extracts were clarified by centrifugation and diluted (2000). accordingly in their respective media supplemented with 50% methanol. 19. Reuter, K., Mofid, M.R., Marahiel, M.A. & Ficner, R. Crystal structure of the sur- The BIA was performed as described12.Briefly,10 µl of supernatant or factin synthetase-activating enzyme Sfp: a prototype of the 4′-phosphopanteth- einyl transferase superfamily. EMBO 18, 6823Ð6831 (1999). extract and twofold serial dilutions thereof were applied to agar plates 20. Kumada, Y. et al. A degradation product of the chromophore of auromomycin. J. seeded with Escherichia coli BR513 and incubated for 3 h at 37 °C. Soft Antibiot. (Tokyo) 36, 200Ð202 (1983).

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