Structure and Bioactivity of a New Furan Fatty Acid from Mumia Sp. YSP-2-79
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J. Gen. Appl. Microbiol., 64, 62–67 (2018) doi 10.2323/jgam.2017.06.004 „2018 Applied Microbiology, Molecular and Cellular Biosciences Research Foundation Full Paper Mumiamicin: Structure and bioactivity of a new furan fatty acid from Mumia sp. YSP-2-79 (Received February 16, 2017; Accepted June 30, 2017; J-STAGE Advance publication date: January 25, 2018) Toru- Kimura,1 Ayano Tajima,2 Yuki Inahashi,3 Masato Iwatsuki,1,3 Hiroaki Kasai,2 Takayuki Mokudai,4 - Yoshimi Niwano,4 Kazuro Shiomi,1,3 Yoko- Takahashi,3 Satoshi Omura,3 and Takuji Nakashima3,* 1 Graduate School of Infection Control Sciences, Kitasato University, Tokyo, Japan 2 School of Marine Biosciences, Kitasato University, Kanagawa, Japan 3 Kitasato Institute for Life Sciences, Kitasato University, Tokyo, Japan 4 Tohoku University Graduate School of Dentistry, Miyagi, Japan tropica CNB-440 (5.1 Mbp) revealed the majority of the A new antibiotic, designated mumiamicin, was iso- 17 known secondary metabolite biosynthetic gene clus- lated from the cultured broth of the rare actino- ters, with a few clusters appearing to encode proteins pre- mycete strain, Mumia sp. YSP-2-79, by Diaion HP- viously identified in other actinobacteria. The biosynthetic 20, silica gel and ODS column chromatography, gene clusters for secondary metabolites constitute approxi- followed by HPLC purification. The chemical mately 10% of the whole genome, which is about twice as structure of mumiamicin was elucidated as a new much as other actinomycetes (Udwary et al., 2007). It is furan fatty acid by nuclear magnetic resonance and expected that there are many untapped marine mass spectrometry. Mumiamicin showed antimi- actinobacteria having the potential to produce unique crobial activity and antioxidative activity. chemicals, making them an invaluable resource for the discovery of novel, useful bioactive compounds. Key Words: antioxidative activity; furan fatty acid; Recently, it has become easy to acquire the UV and MS marine actinomycete; mumiamicin; physicochemi- spectra of many single components from a microbial cul- cal screening tured broth. We have explored cultured broths of microor- ganisms for new compounds using physicochemical (PC) screening, an approach based simply on the physico- Introduction chemical properties of compounds, using LC-UV/vis di- ode array detection (LC/DAD), liquid chromatography Marine habitats and ecospheres differ substantially from combined with mass spectrometry (LC/MS) and simple those in terrestrial environments. Consequently, marine color-change reactions. New compounds, such as the organisms are expected have evolved unique systems, in- spoxazomicins (Inahashi et al., 2011), trehangelins cluding specialized metabolic pathways, for survival un- (Nakashima et al., 2013), mangromicins (Nakashima et der their unique living conditions. In the past decades, al., 2014a, 2014b, 2015), iminimycins (Nakashima et al., many useful natural products, such as salinosporamide 2016a, b), and sagamilactam (Kimura et al., 2016), were (Feling et al., 2003), abyssomicin (Bister et al., 2004) and successfully discovered from metabolites of actinomyc- thallusin (Matsuo et al., 2005), have been discovered from ete strains by our PC screening. marine microorganisms. In particular, salinosporamide A, In our course of PC screening, UV/vis and MS spectra- also known as marizomib, produced by marine guided fractionation of a cultured broth of a marine ac- actinobacteria belonging to the new genus Salinispora, is tinomycete strain, Mumia sp. YSP-2-79, led to the dis- a proteasome inhibitor with promising anticancer activity covery of a new compound, named mumiamicin (1), which against multiple myeloma and is now in Phase I/II clini- consists of fatty acid and furan moieties (Fig. 1). Actino- cal trials (Russo et al., 2015). The genome sequence of S. mycete strain YSP-2-79 was one of the strains isolated *Corresponding author: Takuji Nakashima, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8641, Japan. Tel/Fax: +81-3-5791-6450 E-mail: [email protected] None of the authors of this manuscript has any financial or personal relationship with other people or organizations that could inappropriately influence their work. Mumiamicin: Structure and bioactivity of a new furan fatty acid from Mumia sp. YSP-2-79 63 ¥ 250 mm, GL Sciences Inc., Tokyo, Japan), mobile phase: 5–100% MeOH/0.1% formic acid (linear gradient in 30 min), flow rate: 0.5 mL/min, column temperature: 40∞C, the injection volume: 5 mL, and UV detection: DAD (200– 600 nm). ESI-MS spectra were recorded for 30 min in the m/z region from 100 to 2,000 Da with the following in- strument parameters: ion spray voltage = 5,500 V, source Fig. 1. Structure of mumiamicin (1). gas = 50 L/min, curtain gas = 30 L/min, declustering po- tential = 50 V, focusing potential = 250 V, temperature = 450 ∞C, and detector voltage = 2,300 V. from a sponge sample collected at Japan’s Kagoshima Taxonomic studies of strain YSP-2-79. The 16S rRNA Prefecture. The present paper deals with taxonomic stud- gene sequence was amplified by PCR and sequenced on a ies of the strain, as well as the isolation, physico-chemi- 3130 Genetic Analyzer (Applied Biosystems, Carlsbad, cal properties, structure elucidation, and biological activi- CA, USA) using a BigDye Terminator v.3.1 cycle ties, of mumiamicin (1). sequencing kit (Applied Biosystems), according to the manufacturer’s instructions. Materials and Methods Biological activities. General experimental procedures. All solvents for HPLC Antimicrobial activity and cytotoxicity: Antimicrobial were purchased from Wako Pure Chemical (Osaka, Japan) activities of 1 against six microorganisms, Bacillus subtilis or Kanto Chemical (Tokyo, Japan). NMR spectra were ATCC 6633, Kocuria rhizophila ATCC 9341, Escherichia obtained using XL-400 (Agilent Technologies, Santa coli NIHJ, Xanthomonas campestris pv. oryzae KB 88, Clara, CA, USA) with 1H NMR at 400 MHz and 13C NMR Candida albicans KF1, and Mucor racemosus IFO4581, at 100 MHz in CDCl . Chemical shifts are expressed in were evaluated using the paper disk method (ø6 mm disk, 3 Advantec, Co., Ltd., Tokyo, Japan). Media for microor- ppm and are referenced to residual CDCl3 (7.26 ppm) in 1 13 ganisms were as follows: nutrient agar (Sanko Junyaku, the H NMR spectra and CDCl3 (77.0 ppm) in the C NMR spectra. LC-ESI-MS spectra were obtained using an AB Co., Ltd., Tokyo, Japan) for the bacteria, and for the fungi, Sciex QSTAR Hybrid LC/MS/MS Systems (AB Sciex, the medium was composed of 1.0% glucose, 0.5% yeast Framingham, MA, USA). IR spectra (KBr) were obtained extract and 0.8% agar. A paper disk containing a sample using a Horiba FT-710 Fourier transform IR spectrometer (at a final concentration of 10 mg per disk) was placed on (Horiba, Kyoto, Japan). UV spectra were obtained with a an agar plate with each microorganism. The bacteria, ex- Hitachi U-2810 spectrophotometer (Hitachi, Tokyo, Ja- cept for Xanthomonas campestris pv. oryzae KB 88, were pan). Optical rotation was measured on a JASCO model incubated at 37∞C for 24 h. Fungi and X. campestris pv. DIP-1000 polarimeter (Jasco, Tokyo, Japan). oryzae KB 88 were incubated at 27∞C for 48 h. The cyto- toxic activity of 1 against HeLa S3 and Jurkat cells were Physicochemical screening. A total of 26 actinomycete evaluated, according to previously reported techniques strains isolated from marine sediments and sponges col- (Nakashima et al., 2016a). lected in Japan were used for PC screening. These strains Scavenging effects on hydroxyl radical (·OH) and singlet 1 1 were incubated on a rotary shaker (280 rpm) for 6–14 days oxygen ( O2): Both ·OH and O2 scavenging assays were at 27∞C using 10 mL of four different media in a tube (ø20 essentially identical to those previously described (Katsuda ¥ 250 mm) to prepare 104 cultured broths. The media were et al., 2015). soybean meal medium (3.3% soybean meal, 2.2% soluble For the ·OH scavenging assay, a non-thermal atmos- starch, 2.2% glycerol, 1.2% meat extract, 2.0% peptone, pheric pressure plasma-jet device was used as an ·OH gen- and 2.2% CaCO3), starch medium (2.4% starch, 0.1% glu- erator. The device was connected to a sinusoidal voltage cose, 0.5% peptone, 0.5% yeast extract, 0.3% meat ex- power source with a voltage of 3 kV, and helium gas at a tract, and 0.4% CaCO3), pharmamedia medium (1.0% flow rate of 3 L/min was used as a feeding gas at atmos- pharmamedia, 0.5% glucose, 0.5% corn steep powder, pheric pressure. An aliquot (500 mL) of a reaction mix- 1.0% oatmeal, 0.5% K2HPO4, 0.5% MgSO4·7H2O) and 1 ture comprising 1 mM of 1 and 300 mM 5,5-dimethyl-1- mL/L trace metals solution (0.1% FeSO4·7H2O, 0.1% pyrroline N-oxide (DMPO) (Labotec, Tokyo, Japan) dis- MnCl2·4H2O, 0.1% ZnSO4·7H2O, 0.1% CuSO4·5H2O and solved in pure water was irradiated with the plasma jet for 0.1% CoCl2·6H2O) and defatted wheat germ medium 30 s. DMSO (at a final concentration of 3.5 M) and pure (1.0% defatted wheat germ, 2.0% soluble starch, 0.5% water were used as positive and negative controls, respec- glycerol, 0.3% meat extract, 0.3%, dry yeast, and 0.3% tively. 4-Hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl CaCO3). Each cultured broth was subsequently added with (TEMPOL) (Sigma-Aldrich, Saint Louis, MO, USA) (5 an equal amount of ethanol and then filtered. All filtrates mM) was used as a standard sample to calculate the con- were analyzed by the electrospray ion source of a QSTAR centration of DMPO-OH, and the ESR spectrum of the Elite ESI quadruple time-of-flight (Q-TOF) MS instrument manganese ion, which was equipped in the ESR cavity, (R м 10,000, the tolerance for mass accuracy 10 ppm) was used as an internal standard.