Terrestrial actinobacteria have served as a primary source of bioactive compounds; however, a rapid decrease in the discovery of new compounds strongly necessitates new investigational approaches. One approach is the screening of actinobacteria from marine habitats, especially the members of the genus Streptomyces. Presence of this genus in a marine sponge, Haliclona sp., was investigated using culture-dependent and -independent techniques. 16S rRNA gene clone library analysis showed the presence of diverse Streptomyces in the sponge sample. In addition to the dominant genus Streptomyces, members of six different genera were isolated using four different media. Five phylogenetically new strains, each representing a novel species in the genus Streptomyces were also isolated. Polyphasic study suggesting the classification of two of these strains as novel species is presented. Searching the strains for the production of novel compounds and the presence of biosynthetic genes for secondary metabolites revealed seven novel compounds and biosynthetic genes with unique sequences. In these compounds, JBIR-43 exhibited cytotoxic activity against cancer cell lines. JBIR-34 and -35 were particularly interesting because of their unique chemical skeleton. To our knowledge, this is the first comprehensive study detailing the isolation of actinobacteria from a marine sponge and novel secondary metabolites from these strains.

This article introduces DoBISCUIT (Database of BIoSynthesis clusters CUrated and InTegrated, http://www.bio.nite.go.jp/pks/), a literature-based, manually curated database of gene clusters for secondary metabolite biosynthesis. Bacterial secondary metabolites often show pharmacologically important activities and can serve as lead compounds and/or candidates for drug development. Biosynthesis of each secondary metabolite is catalyzed by a number of enzymes, usually encoded by a gene cluster. Although many scientific papers describe such gene clusters, the gene information is not always described in a comprehensive manner and the related information is rarely integrated. DoBISCUIT integrates the latest literature information and provides standardized gene/module/domain descriptions related to the gene clusters.

A novel tricyclic diterpenoid antibiotic, brasilicardin A, was isolated from the culture broth of Nocardia brasiliensis IFM0406. The antibiotic exhibited immunosuppressive activity in a mouse mixed lymphocyte reaction (MLR)assay system and its IC50 value was 0.057jug/ml. Although the inhibitory activity of cyclosporin A (CyA) against IL-2 production was confirmed in the MLRassay system, brasilicardin A did not have the activity. The results of in vitro toxicity testing of brasilicardin A against various human cell lines were comparedwith those of CyA.During our continuing search for bioactive metabolites from pathogenic Nocardia, we isolated and reported new antibiotics, brasiliquinones A~C1>2), brasilidine A3) and nocardicyclins A, B4). Recently we also reported a new 32-membered macrolide antibiotic, brasilinolide A, with immunosuppressive activity from N. brasiliensis IFM 04065>6). Further studies on the active metabolites from the culture broth of strain IFM 0406 led to the isolation of a new terpenoid antibiotic, brasilicardin A.In this paper we report on the fermentation, isolation and biological activity of brasilicardin A. FermentationThe seed culture was prepared by inoculating mycelial fragments of strain IFM 0406 into a 500 ml-Erlenmeyer flask containing 150ml of brain heart infusion medium (BHI, Difco) supplemented with 1% glucose and 1% glycerol. The culture was performed at 32°C for 96 hours on a rotary shaker at 300rpm. The seed culture (150ml) was then inoculated into a 20 liter jar fermenter containing the production medium (15 liters) consisting of2% glycerol, 1%polypeptone and 0.5% meat extract in distilled water. The pH was adjusted to 7.0 with 1 m NaOHbefore sterilization.Adekanol (Asahi Denka Co., Ltd.) was added as the antifoam. Fermentation was carried out at 32°C for 3 days under aeration of 15 liters/minute and agitation at 200 rpm. HPLCAnalysis of Brasilicardin AThe amount of brasilicardin A in the culture broth was estimated by HPLCanalysis using a reverse phase column (LiChrospher 100 RP-18 endcapped, 4mmx125 mm,Merck). The analysis was carried out as follows:

A new 32-membered macrolide antibiotic, brasilinolide A was isolated from the fermentation broth of Nocardia sp. IFM0406. The producer was identified as Nocardia brasiliensis. The antibiotic was only active against Aspergillus niger, but not active against other fungi including yeasts as well as other filamentous like fungi and bacteria. Brasilinolide A exerted an immunosuppressive activity in the assay system of a mixed lymphocyte reaction (MLR). Immunosuppressive agents which attack specific target sites of immunocompetentcells are good candidate for immunotherapy. The examples of useful drugs of this type are cyclosporin A1} and FK-5062). We had reported that some opportunistic pathogenic Nocardia, such as Nocardia otitidiscaviarum and Nocardia brasiliensis, produce novel bioactive substances3~5). In the course of continuing search for bioactive substances including immunosuppressive agents from Nocardia, we found

A novel tricyclic metabolite, brasilicardin A (1), with potent immunosuppressive activity has been isolated from the cultured broth of the actinomycete Nocardia brasiliensis IFM 0406, and the structure including absolute stereochemistry was established on the basis of spectroscopic data, chemical means, and X-ray analysis. Brasilicardin A (1) is the first tricyclic compound consisting of an anti/syn/anti-perhydrophenanthrene skeleton with a rhamnose, an N-acetylglucosamine, and an amino acid moiety.

To identify the species of butyrolactol-producing Streptomyces strain TP-A0882, whole genome-sequencing of three type strains in a close taxonomic relationship was performed. In silico DNA-DNA hybridization using the genome sequences suggested that Streptomyces sp. TP-A0882 is classified as Streptomyces diastaticus subsp. ardesiacus. Strain TP-A0882, S. diastaticus subsp. ardesiacus NBRC 15402T, Streptomyces coelicoflavus NBRC 15399T, and Streptomyces rubrogriseus NBRC 15455T harbor at least 14, 14, 10, and 12 biosynthetic gene clusters (BGCs), respectively, coding for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). All 14 gene clusters were shared by S. diastaticus subsp. ardesiacus strains TP-A0882 and NBRC 15402T, while only four gene clusters were shared by the three distinct species. Although BGCs for bacteriocin, ectoine, indole, melanine, siderophores such as deferrioxamine, terpenes such as albaflavenone, hopene, carotenoid and geosmin are shared by the three species, many BGCs for secondary metabolites such as butyrolactone, lantipeptides, oligosaccharide, some terpenes are species-specific. These results indicate the possibility that strains belonging to the same species possess the same set of secondary metabolite-biosynthetic pathways, whereas strains belonging to distinct species have species-specific pathways, in addition to some common pathways, even if the strains are taxonomically close.

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