Characterization of Surfactin-like Cyclic Depsipeptides Synthesized by Bacillus pumilus from Ascidian Halocynthia aurantium Natalie I. Kalinovskaya,1* Tatyana A. Kuznetsova,1 Elena P. Ivanova,1** Ludmila A. Romanenko,1 Valery G. Voinov,1 Felix Huth,2 and Hartmut Laatsch2 1Pacific Institute of Bio-organic Chemistry of the Far-Eastern Branch of the Russian Academy of Sciences, 159 pr. 100-Let Vladivostoku, 690022 Vladivostok, Russian Federation 2Department of Organic Chemistry, University of Goettingen, Tammanstr. 2. D-37077 Goettingen. Germany * Corresponding author; telephone: +7(423 2)311168; fax: +7(423 2)314050; e-mail: [email protected] ** Present address: Swinburne University of Technology, 533-545 Burwood Rd, Hawthorn, 33122, Melbourne, Australia Running title: Surfactin-like cyclic depsipeptides from Bacillus pumilus Key words: Surfactin analog, cyclic depsipeptides, Bacillus pumilus, ascidian Halocynthia aurantium Summary: A marine bacterium (KMM 1364), identified as Bacillus pumilus, was isolated from the surface of ascidian Halocynthia aurantium. Structural analysis revealed that the strain KMM 1364 produced a mixture of lipopeptide surfactin analogs with major components ranging in size from molecular masses of 1035, 1049, 1063 and 1077. The variation in molecular weight represents changes in the number of methylene groups in the lipid and/or peptide portion of the compounds. Structurally, these lipopeptides differ from surfactin in the subsitution of the valine residue in position 4 by leucine, and have been isolated as two C-terminal variants, with valine or isoleucine in position 7. As constituents of the lipophilic part of the peptides, only β-hydroxy-C15-, β-hydroxy-C16- and a high amount of β-hydroxy-C17 fatty acid were determined. INTRODUCTION Biosurfactants comprise a structurally diverse group of surface-active compounds produced by microorganisms. Increasing interest to these substances is explained by the wide range of their biotechnological applications including enhanced oil recovery due to reduction of surface and interfacial tensions in aqueous solution and hydrocarbon mixtures (Singer, 1985), deemulsification (Cairns et al., 1982; Mulligan, and Gibbs, 1993), health care (Muller- Hurting et al., 1993), and food-processing industries (Velikonja and Kosaric, 1993). Microbial surfactants are of particular interest because they, in contrast to synthetic analogs, are biodegradable and can be produced through fermentation on renewable substrates. The biosurfactant-producing microorganisms belong to different taxa (Desai and Banat, 1997). However, among Gram-positive spore-forming bacteria only a few species of the genus Bacillus (i.e. B. subtilis and B. licheniformis), Brevibacillus brevis (former Bacillus brevis) and Paenibacillus polymyxa (former Bacillus polymyxa) are known producers of cyclic lipopeptides including decapeptide antibiotics (gramicidins) and lipopeptides antibiotics (polymyxins) with surface-active and chelating properties. The cyclic lipopeptide surfactin from B. subtilis was intensively structurally studied in respect to its biotechnological and pharmacological applications (Cooper et al., 1981; Vollenbroich et al., 1997). A surfactin like lipopeptide surfactant, lichenisin, is produced by B. licheniformis JF-2 and was patented as an enhanced oil recowery agent (McInerney et al., 1985). The structure, molecular genetics, properties, and production of biosufactants have been reviewed recently (Desai and Banat, 1997; Sullivan, 1998). Over the last decade we studied the biologically active secondary metabolites produced by marine bacteria isolated from both seawater and an array of marine invertebrates (Elyakov et al., 1996). Previously we reported about cyclic depsipeptides found in B. pumilus isolated from the sponge Ircinia sp. (Kalinovskaya et al., 1995). This paper describes the isolation and structure elucidation of one of the isoforms of surfactin produced by B. pumilus, isolated from surface of cuticula of the ascidian Halocynthia aurantium, common inhabitant of the Sea of Japan. MATERIALS AND METHODS Instruments UV spectra were recorded on a Specord UV-VIS M 40 spectrophotometer (Carl Zess Jena) in methanol. IR spectra were obtained on a Specord M-82 (Carl Zeiss Jena) spectrometer in CHCl3. The amino acid composition was determined on an amino acid analyzer (Biotronic LC 2000, Sweden, using DS-6A resin column) after total hydrolysis of the lipopeptides in 5.6 N HCl at 105oC for 48 h. NMR spectra were recorded on a Bruker AM 500 spectrometer (500 and 125.7 MHz for 1H and 13C, respectively, using TMS as the internal reference). The native peptides were analyzed by fast atom bombardment mass spectrometry (FABMS) on a Finnigan 95 A mass spectrometer, using a 3-nitro-benzaldehyde matrix (3-NBA). Positive and negative ions were detected. Bacterial strain The strain Bacillus pumilus KMM 1364 was isolated from the ascidian Halocynthia aurantium in August 1989, the Troitza Bay, Gulf of Peter the Great (Romanenko et al., 2001). This strain was grown on a rotary shaker (120 rpm) in 1 L Erlenmeyer flasks containing 200 ml of the medium (g/L): K2HPO4 (0.07), NH4Cl (1.0 ), yeast extract (5.0 ), FeSO4 (0.025 ), 1 M Tris buffer (20 ml), artificial sea water (200 ml), distilled water (800 ml), pH adjusted to 7.5, during 20 h at 24-26oC. Isolation of Depsipeptides 1, 2, 6-8 The cells (from 6 L of culture medium) were collected by centrifugation at 5000 g for 40 min and suspended in water (50 ml), frozen and destructed by ultrasonic treatment. The suspension was extracted with a mixture of chloroform-methanol (3:1) and the organic phase evaporated to dryness. Column chromatography of the сrude extract (402 mg) on a silica gel column (40/100 µ, Chemapol, Czechoslovakia, 1.5x20 cm) with solvents of increasing polarity gave the following fractions: hexane-ethyl acetate 3:1 (F1-F4); 2:1 (F5-F7); 1:1 (F8-F9); 1:3 (F10-F11); ethyl acetate (F12-F14); ethyl acetate-MeOH, 95:5 (F15-F17); 9:1 (F18-F19); 1:1 (F20-F21). Thin-layer Chromatography (TLC) The mixture of peptides was analyzed by TLC on silica gel 5/40 µ (Czechoslovakia) in a chloroform-methanol-water mixture (65:25:4, v/v/v), the zones were visualized using the chlorine-tolidine reaction. High Pressure Liquid Chromatography (HPLC) The combined fractions F10-F17 were separated by reversed phase HPLC with a Waters instrument (USA) on a Separon SGX column (C18, 5 µ, 4.0 i. d. x 250 mm, acetonitrile-0.01% TFA/H2O 86:14, flow rate 1.5 ml/min, detection by UV absorption at 214 nm). The individual depsipeptides 1, 2, and 6-8 were obtained at retention times of t1=16.2, t2=20.7, t6/7=29.7, and t8=37.6 min. NMR Data of Peptide 1 ([D6]-Acetone) 13С NMR: 173.90 (Cδ), 173.90 (CO), 172.15 (2-CO), 171.74 (CO), 171.60 (CO), 171.14 (CO), 170.66 (Cγ), 170.25 (CO), 57.45 (α-CH), 52.40 (α-CH), 51.75 (α-CH), 51.34 (α-CH), 50.83 (α- CH), 49.81 (α-CH), 48.54 (α-CH), 40.68 (β-CH2), 39.0 (β-CH2), 38.43 (2 β-CH2), 35.95 (β- CH2), 31.23 (β-CH2), 29.36 (β-CH), 28.57 (γ-CH2), 27.32 (γ-CH), 24.22 (γ-CH), 24.20 (γ-CH), 24.09 (γ-CH), 22.92 (δ1-CH3), 22.88 (δ2-CH3), 22.81 (δ1-CH3), 22.64 (δ2-CH3), 21.78 (δ1-CH3), 21.63 (δ2-CH3), 21.44 (δ1-CH3), 21.39 (δ2-CH3), 19.01 (γ1-CH3), 18.1 (γ2-CH3); alkyl chain: 169.63 (C-1), 41.06 (C-2, CH2), 71.72 (C-3, CH), 33.42 (C-4, CH2), 26.70 (C-5, CH2), 29.24- 28.82 (C-6-C-10, CH2), 30.04 (iso, C-11, CH2), 29.24 (anteiso, C-11, CH2), 38.81 (iso, C-12, CH2), 33.69 (anteiso, C-12, CH), 27.32 (iso, C-13, CH), 29.24 (anteiso, C-13, CH2), 22.42 (iso, 1 C-14, CH3), 11.09 (anteiso, C-14, CH3), 22.42 (iso, 13-Met), 19.16 (anteiso, 12-Met). H NMR: 4.71, 4.52, 4.37, 4.25, 4.12, 4.06 (2H, αCH), 2.49 t, 2.28, 1.60-1.46 (8H), 2.88 d, 2.49, 2.64 d (βCH); 1.88-1.60 (4H), 1.60-1.46 (2H, γCH); 1.02-0.8 (10 CH3); alkyl chain: 2.69, 2.15 (C-2, H), 5.33 (C-3, H), 1.60-1.46 (C-4, H), 1.30-1.15 (CH2)5-12, 1.15 (C-13), 1.02-0.8 (C-14-15). NH: 8.14 (d, J=6.0), 7.86 (d, J=7.2), 7.79 (t, 2H, J=7.2), 7.72 (d, J=8.5), 7.60 (d, J=6.0), 7.34 (d, J=8.0). NMR Data of Peptide 2 ([D6]-DMSO) 13С NMR: 173.89 ( CO), 173.89 (Cδ),-172.06 (2 CO), 171.63 (Cγ), 171.55 (CO), 171.09 (CO), 170.61 (CO), 170.13 (CO), 56.43 (α-CH), 52.29 (α-CH), 51.78 (α-CH), 51.71 (α-CH), 51.30 (α-CH), 50.74 (α-CH), 49.77 (α-CH), 40.55 (β-CH2), 39.44 (β-CH2), 38.39 (2 β-CH2), 35.91 (β- CH2), 35.74 (β-CH), 30.00 (β-CH2), 27.28 (γ-CH), 27.12 (γ-CH2), 24.50 (γ1-CH2), 24.19 (γ-CH), 24.16 (γ-CH), 24.07 (γ-CH), 22.89 (δ1-CH3), 22.86 (δ2-CH3), 22.81 (CH3), 22.61 (CH3), 21.81 ((δ1-CH3), 21.62 (δ2-CH3), 21.44 (δ1-CH3), 21.31 (δ2-CH3), 15.41 (γ2-CH3), 10.95 (δ-CH3); alkyl chain: 169.54 (C-1), 41.05 (C-2, CH2), 71.74 (C-3, CH), 33.26 (C-4, CH2), 26.66 (С-5, CH2), 29.20-28.78 [(C-6-C-10), CH2], 29.20 (iso, C-11, CH2), 28.52 (anteiso, C-11, CH2), 39.00 (iso, C-12, CH2), 33.66 (anteiso, C-12, CH), 27.28 (iso, C-13, CH), 29.26 (anteiso, C-13, CH2), 22.39 1 (iso, C-14, CH3), 11.07 (anteiso, C-14, CH3), 22.39 (iso, 13-Met), 18.98 (anteiso, 12-Met).