Genomic analyses of Amycolatopsis orientalis ATCC 43491 strain, deposited as a vancomycin producer, revealed the presence of genetic loci for the production of at least 10 secondary metabolites other than vancomycin. One of these gene clusters, which contained a type I polyketide synthase, was predicted to direct the synthesis of novel class of compound, a glycosidic polyketide ECO-0501 (1). Screening of culture extracts for a compound with the predicted physicochemical properties of the product from this locus, led to the isolation of the 13-Oglucuronide of 13-hydroxy-2,12,14,16,22-pentamethyl-28-(N-methyl-guanidino)-octacosa-2,4,6,8,10,14,20,24-octaenoic acid (2-hydroxy-5-oxo-cyclopent-1-enyl)-amide (ECO-0501, 1). The structure, confirmed by spectral analyses including MS, and 1D and 2D NMR experiments, were in accord with that predicted by genomic analyses. ECO-0501 possessed strong antibacterial activity against a series of Gram-positive pathogens including several strains of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococci (VRE). ECO-0501 was chemically modified by esterification (1aϳ1c), Nacetylation (1d) and hydrogenation (1e) in order to explore structure activity relationships (SAR). Keywords Amycolatopsis orientalis, ECO-0501, antibacterial, PKS I IntroductionDrug-resistant bacterial infections are a growing health concern. Resistance has been developed to every major class of antibiotics on the market, and an increasing number of pathogenic bacteria are becoming resistant to multiple classes of antibiotics, thereby limiting treatment options. Hence, there is a renewed urgency for the discovery of new classes of antibiotics for the treatment of drug resistant bacterial infections. To accelerate the discovery of such potential antibacterial candidates from natural resources a new, fast and efficient technology is needed. The genomics of secondary metabolite biosynthesis recently evolved to the point where analysis of the genome of an organism can define its biosynthetic capabilities for secondary metabolites. A genome scanning technique that has been developed in our laboratories, and used with our DECIPHER ® technology to analyze the genomes of actinomycetes for their secondary metabolite biosynthetic genes, greatly reduces the amount of sequencing required to define this capability [1,2]. This approach not only ascertains the potential of a producing organism, but it provides a handle to detect, isolate and structurally define a specific metabolite. We have demonstrated this approach in the isolation and structural determination of an antifungal Genomic Analyses Lead to Novel Secondary MetabolitesPart 3

The novel microbial metabolite diazepinomicin/ECO-4601 (1) has a unique tricyclic dibenzodiazepinone core, which was unprecedented among microbial metabolites. Labeled feeding experiments indicated that the carbocyclic ring and the ring nitrogen of tryptophan could be incorporated via degradation to the 3-hydroxyanthranilic acid, forming ring A and the nonamide nitrogen of 1. Genomic analysis of the biosynthetic locus indicated that the farnesyl side chain was mevalonate derived, the 3-hydroxyanthranilic acid moiety could be formed directly from chorismate, and the third ring was constructed via 3-amino-5-hydroxybenzoic acid. Successful incorporation of 4,6-D2-3-hydroxyanthranilic acid into ring A of 1 via feeding experiments supports the genetic analysis and the allocation of the locus to this biosynthesis. These studies highlight the enzymatic complexity needed to produce this structural type, which is rare in nature.

Analyses of biosynthetic gene clusters derived from Streptomyces aculeolatus NRRL 18422 and Streptomyces sp. Eco86 indicated that both microorganisms have similar type I polyketide synthase (PKS) gene clusters with relatively few genes encoding post-PKS elaborative enzymes. However both gene clusters included a sequence coding for a relatively uncommon oxidative enzyme related to Baeyer-Villiger, flavin-type monooxygenases. Screening of culture extracts for compounds with the predicted physicochemical properties of the end products from these loci, led to the isolation of three 5-alkenyl-3,3(2H)-furanones, one (E-837, 1) from the former and two (E-492, 2, E-975, 3) from the latter strain. The structures, confirmed by spectral analyses including MS, and 1D and 2D NMR experiments, were in accord with those predicted by genomic analyses. Baeyer-Villiger type oxidation is postulated to be involved in the formation of the furanone moieties in these molecules. All three new compounds were tested for their electron transport inhibitory activities. They had IC 50 values of 1ϳ4 mg/ml against Ascaris suum NADH-fumarate reductase and 1ϳ12 mg/ml against bovine heart NADH oxidase.Keywords Streptomyces aculeolatus, Streptomyces sp., E-837, E-492, E-975, Baeyer-Villiger monooxygenase IntroductionNatural products play an important role in drug discovery and have been used for the treatment of various diseases for decades. They constitute a leading source of novel molecules for the development of new drug candidates to treat life threatening infections and other human disorders [1]. To identify such potential drug candidates from nature, different methods have been developed and routinely used in natural product discovery laboratories. The genomics of secondary metabolite biosynthesis has recently evolved to the point where analysis of the genome of an organism can define its secondary metabolic capabilities. A genome scanning technique has been developed in our laboratories to greatly reduce the amount of sequencing required to define this capability [2,3]. This approach not only ascertains the potential of a producing organism, but it provides the scientist with a handle to identify, isolate and structurally define a specific metabolite. We have demonstrated this approach in the isolation and structural determination of an antifungal agent, ECO-02301 from Streptomyces aizunensis [4].In our continuing effort to find novel secondary metabolites from actinomycetes, we observed that two different streptomycetes, Streptomyces aculeolatus NRRL 18422 (a producer of the semi-naphthaquinone antibiotic, [5] and Streptomyces sp. Eco86 (a proprietary Ecopia strain) had similar gene clusters for type I polyketides with relatively few post-polyketide elaborative enzymes, but including an uncommon Baeyer-Villiger type monooxygenase. Analyses of these gene clusters led us to conclude that these microorganisms produce members of a group of very closely related secondary metabolites. Here, we report the isolation and identification of...

The deposited strain of the hazimicin producer, Micromonospora echinospora ssp. challisensis NRRL 12255 has considerable biosynthetic capabilities as revealed by genome scanning. Among these is a locus containing both type I and type II PKS genes. The presumed products of this locus, TLN-05220 (1) and TLN-05223 (2), bear a core backbone composed of six fused rings starting with a 2-pyridone moiety. The structures were confirmed by conventional spectral analyses including MS, and 1D and 2D NMR experiments. Comparison of both the 1 H and 13 C NMR data of the newly isolated compound with those of echinosporamicin and bravomicin A led us to propose a revision of the structure of the latter to include a 2-pyridone instead of the pyran originally postulated. Both compounds (1 and 2) possessed strong antibacterial activity against a series of grampositive pathogens including several strains of methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococci (VRE), and cytotoxic activities against several human tumor cell lines. The TLN compounds are the first of this group with reported anticancer activity.

Isolation and Identification of Three New 5-Alkenyl-3,3(2H)-furanones from Two Streptomyces Species Using a Genomic Screening Approach. -E-837(Ia), E-492 (Ib), and E-975 (Ic) are tested for their electron transport enzyme inhibition. Compared with nafuredin, (I) appear to be non-selective electron transport complex inhibitors of moderate potency. -(BANSKOTA, A. H.; MCALPINE*, J.; SOERENSEN, D.; AOUIDATE, M.; PIRAEE, M.; ALARCO, A.-M.; OMURA, S.; SHIOMI, K.; FARNET, C. M.; ZAZOPOULOS, E.; J. Antibiot. 59 (2006) 3, 168-176; Ecopia BioSciences Inc., Montreal, Que. H4S 2A1, Can.; Eng.) -Schulze 39-222