The enediynes exemplify nature's ingenuity. We have cloned and characterized the biosynthetic locus coding for perhaps the most notorious member of the nonchromoprotein enediyne family, calicheamicin. This gene cluster contains an unusual polyketide synthase (PKS) that is demonstrated to be essential for enediyne biosynthesis. Comparison of the calicheamicin locus with the locus encoding the chromoprotein enediyne C-1027 reveals that the enediyne PKS is highly conserved among these distinct enediyne families. Contrary to previous hypotheses, this suggests that the chromoprotein and nonchromoprotein enediynes are generated by similar biosynthetic pathways.

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five 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). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or "warhead") found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.

Mutations in the DAX-1 gene are responsible for congenital X-linked adrenal hypoplasia, a disease that is associated with hypogonadotropic hypogonadism. DAX-1 expression is tissue-specific and is finely regulated throughout development, suggesting that it has a role in both adrenal and gonadal function. DAX-1 is an unusual member of the nuclear-receptor superfamily of transcription factors which contains no canonical zinc-finger or any other known DNA-binding motif. Binding sites for DAX-1 are found in the promoters of the dax-1 and StAR (for steroidogenic acute regulatory protein) genes. Here we show that DAX-1 binds DNA and acts as a powerful transcriptional repressor of StAR gene expression, leading to a drastic decrease in steroid production. We provide in vitro and in vivo evidence that DAX-1 binds to DNA hairpin structures. Our results establish DAX-1 as the first member of the nuclear receptor superfamily with novel DNA-binding features and reveal that it has regulatory properties critical to the understanding of its physiological functions.

Analysis of the genome of Streptomyces aizunensis NRRL B-11277 indicated its potential to produce a compound of novel and highly predictable structure. The structure was predicted with sufficient accuracy to allow straightforward detection of the specific metabolite in HPLC profiles of fermentation extracts and hence to guide the isolation. The spectroscopic work was reduced to a confirmation of structure rather than a first principle determination. The compound, ECO-02301 (1), demonstrated potent antifungal activity. This work exemplifies not only the discovery of novel antibiotics from well-characterized organisms but also the utility of genomics as a further tool, complementary to spectroscopy, to enable rapid determination of complex structures.

The biosynthetic gene cluster for the enediyne antitumor antibiotic neocarzinostatin (NCS) was localized to 130 kb continuous DNA from Streptomyces carzinostaticus ATCC15944 and confirmed by gene inactivation. DNA sequence analysis of 92 kb of the cloned region revealed 68 open reading frames (ORFs), 47 of which were determined to constitute the NCS cluster. Sequence analysis of the genes within the NCS cluster suggested dNDP-D-mannose as a precursor for the deoxy aminosugar, revealed two distinct type I polyketide synthases (PKSs), and supported a convergent model for NCS chromophore biosynthesis from the deoxy aminosugar, naphthoic acid, and enediyne core building blocks. These findings shed light into deoxysugar biosynthesis, further support the iterative type I PKS paradigm for enediyne core biosynthesis, and unveil a mechanism for microbial polycyclic aromatic polyketide biosynthesis by an iterative type I PKS.

Structural inspection of the bacterial meroterpenoid antibiotics belonging to the napyradiomycin family of chlorinated dihydroquinones suggests that the biosynthetic cyclization of their terpenoid subunits is initiated via a chloronium ion. The vanadium-dependent haloperoxidases that catalyze such reactions are distributed in fungi and marine algae and have yet to be characterized from bacteria. The cloning and sequence analysis of the 43-kb napyradiomycin biosynthetic cluster (nap) from Streptomyces aculeolatus NRRL 18422 and from the undescribed marine sediment-derived Streptomyces sp. CNQ-525 revealed 33 open reading frames, three of which putatively encode vanadium-dependent chloroperoxidases. Heterologous expression of the CNQ-525-based nap biosynthetic cluster in Streptomyces albus produced at least seven napyradiomycins, including the new analog 2-deschloro-2-hydroxy-A80915C. These data not only revealed the molecular basis behind the biosynthesis of these novel meroterpenoid natural products but also resulted in the first in vivo verification of vanadium-dependent haloperoxidases.Nature has devised several mechanisms to polarize the terminal olefin of linear terpenes to facilitate the creation of new C-X bonds. For instance, cyclization of the C 30 hydrocarbon squalene to steroids and hopanoids is initiated, respectively, by epoxidation or protonation of the terminal olefin. Although these biosynthetic strategies are widely distributed, a third mechanism for terpene cyclization has been characterized in marine macroalgae involving bromonium ion-induced ring closure (1-3). Oxidation of the halide is catalyzed by vanadiumdependent bromoperoxidase in the presence of hydrogen peroxide to produce the corresponding hypohalous acid. This species then further reacts with electron-rich organic substrates in a regio-and stereoselective manner, giving rise to brominated terpenes and other halogenated natural products (1, 2, 4). Vanadium-dependent bromoperoxidases are widely distributed in marine algae, and the first enzyme was discovered in 1984 from the brown alga Ascophyllum nodosum (5).Vanadium chloroperoxidases (V-ClPOs), 3 on the other hand, have been isolated primarily from dematiaceous hyphomycete fungi (1). The first enzyme was characterized in 1993 from Curvularia inaequalis (6), and even though there are numerous chlorinated marine natural products, V-ClPOs have not been reported from marine organisms to date (1, 2, 7). Although the biological function of V-ClPOs has not yet been elucidated, marine algal vanadium-dependent bromoperoxidases have been shown through in vitro chemoenzymatic conversions to catalyze bromonium ion-initiated cyclization of terpenes and ethers (1,8). These studies not only demonstrated that the enzymes were able to initiate cyclization of a terpene by a bromonium ion but also proved that the halogenation reaction occurred with stereochemical control. To date, all known V-dependent haloperoxidases have been characterized in vitro from eukaryotic systems (9).Structural inspection of...

Anthramycin is a benzodiazepine alkaloid with potent antitumor and antibiotic activity produced by the thermophilic actinomycete Streptomyces refuineus sbsp. thermotolerans. In this study, the complete 32.5 kb gene cluster for the biosynthesis of anthramycin was identified by using a genome-scanning approach, and cluster boundaries were estimated via comparative genomics. A lambda-RED-mediated gene-replacement system was developed to provide supporting evidence for critical biosynthetic genes and to validate the boundaries of the proposed anthramycin gene cluster. Sequence analysis reveals that the 25 open reading frame anthramycin cluster contains genes consistent with the biosynthesis of the two halves of anthramycin: 4 methyl-3-hydroxyanthranilic acid and a "dehydroproline acrylamide" moiety. These nonproteinogenic amino acid precursors are condensed by a two-module nonribosomal peptide synthetase (NRPS) terminated by a reductase domain, consistent with the final hemiaminal oxidation state of anthramycin.

iso-Migrastatin and related glutarimide-containing polyketides are potent inhibitors of tumor cell migration and their implied potential as antimetastatic agents for human cancers has garnered significant attention. Genome scanning of Streptomyces platensis NRRL 18993 unveiled two candidate gene clusters (088D and mgs); each encodes acyltransferase-less type I polyketide synthases commensurate with iso-migrastatin biosynthesis. Both clusters were inactivated by -RED-mediated PCR-targeting mutagenesis in S. platensis; iso-migrastatin production was completely abolished in the ⌬mgsF mutant SB11012 strain, whereas inactivation of 088D-orf7 yielded the SB11006 strain that exhibited no discernible change in iso-migrastatin biosynthesis. These data indicate that iso-migrastatin production is governed by the mgs cluster. Systematic gene inactivation allowed determination of the precise boundaries of the mgs cluster and the essentiality of the genes within the mgs cluster in iso-migrastatin production. The mgs cluster consists of 11 open reading frames that encode three acyltransferase-less type I polyketide synthases (MgsEFG), one discrete acyltransferase (MgsH), a type II thioesterase (MgsB), three post-PKS tailoring enzymes (MgsIJK), two glutarimide biosynthesis enzymes (MgsCD), and one regulatory protein (MgsA). A model for isomigrastatin biosynthesis is proposed based on functional assignments derived from bioinformatics and is further supported by the results of in vivo gene inactivation experiments.Cell migration is essential for invasion of the extracellular matrix and for cell dissemination during tumor metastasis (1). The glutarimide-containing polyketides iso-migrastatin (iso-MGS), 2 migrastatin (MGS), the dorrigocins (DGNs), and lactimidomycin (LTM) (Fig. 1) are potent inhibitors of human tumor cell migration and thus represent novel leads for anticancer drug discovery (2-5). Synthetic analogs of these natural products have also been investigated and found to retain potent activity despite significant structural truncation (6 -8). Retention of activity by such analogs supports the effectiveness of the privileged scaffolds highlighted by iso-MGS, MGS, LTM, and related natural products. Complementary to organic synthesis, combinatorial biosynthesis offers an alternative means of accessing natural product structural diversity. We have previously studied the biosynthetic pathway of these polyketides and shown that MGS and the DGNs are shunt metabolites of iso-MGS (9). As has been demonstrated in Streptomyces platensis NRRL 18993, one of the known iso-MGS producers, iso-MGS, MGS, and the DGNs are produced by a single biosynthetic machinery and iso-MGS undergoes H 2 O-mediated, non-enzymatic ring-expansion, and ring-opening rearrangements to afford MGS and the DGNs as shunt metabolites. We have also isolated iso-MGS congeners as minor fermentation products, produced a small library of glutarimide-containing polyketides featuring the iso-MGS, LTM, MGS, and DGN scaffolds, and found selected analogs with biological a...