JBIR-65, a new diterpene, isolated from a sponge-derived Actinomadura sp. SpB081030SC-15

Abstract

Marine microorganisms, particularly, marine actinomycetes, are one of the most important resources for new biologically active metabolites, 1 and in fact, many new compounds have been isolated from sponge-derived actinomycetes. [2][3][4] We have recently discovered novel compounds, namely the anthracyclines tetracenoquinocin and 5-iminoaranciamycin, 5 a teleocidin JBIR-31, 6 the tetrapeptides JBIR-34 and JBIR-35, 7 the isoprenoids JBIR-46, JBIR-47, JBIR48 8,9 and a new salicylamide JBIR-58. 10 Our intention was to support the idea that new species are capable of producing unique metabolites. For this purpose, we isolated new species of actinomycetes from marine sponges and then searched for secondary metabolites in the culture of isolated strains. In this study, we isolated a new species (SpB081030SC-15) of Actinomadura from an unidentified marine sponge and purified a new diterpene compound designated as JBIR-65 (1, Figure 1a) from the fermentation broth of Actinomadura sp. SpB081030SC-15. In this study, we report the fermentation procedure to obtain 1 and its subsequent isolation, structure elucidation and, in brief, its biological activities.Actinomadura sp. SpB081030SC-15 was isolated from an unidentified marine sponge collected offshore of Ishigaki Island, Okinawa Prefecture, Japan. To identify the species of the strain SpB081030SC-15, we compared its 16S ribosomal RNA gene sequences (Accession No. AB123566) with those available in the DNA Data Bank of Japan using a basic local alignment search tool. 11 The strain was identified as a new species of the genus Actinomadura because comparison of its 16S ribosomal RNA gene sequence revealed a low sequence similarity of 98% with Actinomadura nitritigenes (AB364595).The strain SpB081030SC-15 was cultivated in 50-ml test tubes each containing 15 ml of a seed medium consisting of starch (Kosokagaku, Tokyo, Japan) 1.0%, polypeptone (Nihon Pharmaceutical, Tokyo, Japan) 1.0%, molasses (Dai-Nippon Meiji Sugar, Tokyo, Japan) 1.0% and meat extract 1.0% (Extract Ehlrich, Wako Pure Chemical Industry, Osaka, Japan), pH 7.2 (adjusted before sterilization). The seed culture in test tubes was agitated on a reciprocal shaker (355 r.p.m.) at 27 1C for 2 days. Aliquots (2.5 ml) of the broth were transferred to 500-ml baffled Erlenmeyer flasks containing 100 ml of a production medium consisting of glycerin 2.0%, molasses 1.0%, casein 0.5% and CaCO 3 0.4%, pH 7.2 (adjusted before sterilization) and cultured on a rotary shaker (180 r.p.m.) at 27 1C for 5 days.The mycelia in the fermentation broth (2 l) was separated by centrifugation and then extracted with 80% acetone. The extract was evaporated in vacuo to remove the acetone, and the aqueous residue was extracted with EtOAc. The organic layer was dried over Na 2 SO 4 and evaporated to dryness. The residue (700 mg) was subjected to normal-phase medium-pressure liquid chromatography (Purif-Pack SI-60, Moritex, Tokyo, Japan) and eluted with a gradient system of n-hexane-EtOAc (0-30% EtOAc) and CHCl 3 -MeOH (0-50% MeOH), successively....

Bacterial diterpene synthases: new opportunities for mechanistic enzymology and engineered biosynthesis

“…K04-0144 [38], the gifhornenolones from Verrucosispora gifhornesis YM28-088 [39], and JBIR-65 from Actinomadura sp. SpB081030SC-15 [40]. The actinomycetes have emerged as prolific producers of bacterial diterpenoids [1,2].…”

Natural Products of Actinobacteria Derived from Marine Organisms

“…It contains an unusual natural phosphoramide methyl ester, which was weakly inhibited the acetylcholinesterase in in vitro studies [114]. showed the modest radical scavenging activity [115]. showed the modest radical scavenging activity [115].…”

“…CP32 associated with Conus pulicarius produced five new metabolites, pulicatins A-E (108-112) along with five known analogs (113)(114)(115)(116)(117). CP32 associated with Conus pulicarius produced five new metabolites, pulicatins A-E (108-112) along with five known analogs (113)(114)(115)(116)(117).…”

Actinomadura montaniterrae sp. nov., isolated from mountain soil

“…In addition, many strains are found in other ecosystems such as marine sources (Ara et al, 2008;He et al, 2012) and plant tissues (Qin et al, 2009(Qin et al, , 2012. With respect to drug discovery, the members of this genus produce many types of bioactive metabolites, including alkaloid staurosporine analog (Han et al, 2005;Shaaban et al, 2015), diterpene (Takagi et al, 2010), sterol bendigols (Simmons et al, 2011), siderophore madurastatin C1 (Mazzei et al, 2012), polyketide nomimicin (Igarashi et al, 2012), b-carbiline (Kornsakulkarn et al, 2013) and 3-oxyanthranilic acid (Intaraudom et al, 2014). During the course of an investigation of rare actinomycetes found in soils in Thailand, an actinomycete strain, CYP1-1B T , was isolated from mountain soil.…”