Strains and culture conditions
All strains and plasmids used and constructed in this study are listed in Table S1. Streptomyces sp. SCSIO 01127[9] was cultured at 28 ºC in 38# medium (yeast extract 4 g L-1, glucose 4 g L-1, maltose 5 g L-1multi-vitamins 500 μL L-1, sea salt 30 g L-1, agar powder 15-20 g L-1, pH 7.0 -7.5), and the heterologous recombinant host Streptomyces coelicolor M1154/pCSG5560[13] and the derivatives were cultured at 28 ºC in SFM medium (soybean powder 20 g L-1, mannitol 20 g L-1, agar powder 15-20 g L-1) for growth and sporulation.Escherichia coli strains were grown in Luria-Bertani medium at 37 °C.
DNA isolation, manipulation and sequencing
DNA isolation and manipulation in E. coli and actinomycetes were carried out according to standard procedures.[26, 27] Primers used in this study (Table S2) were synthesized at the Shanghai Invitrogen Biotech Co., Ltd. DNA sequencing was performed at the Invitrogen Biotech Co., Ltd. (Guangzhou), and Chinese National Genome Center (Shanghai).
Disruption of lobP1
The lambda-RED-mediated gene replacement was performed as a standard procedure.[28] The gene disruption experiments inStreptomyces sp. SCSIO 01127 were carried out using the previously reported genetic manipulation system.[9] Details for lobP1 -gene disruption in native strain Streptomyces sp. SCSIO 01127 were described in Figure S1. The disruption of lobP1 in heterologous host S. coelicolor M1154 was conducted using the previously reported genetic manipulation system.[13] Details for the construction of ∆lobP1 mutant S. coelicolorM1154/pCSG5661 were described in Figure S8.
Fermentation and metabolite analysis
The fermentation of all strains in this study were carried out using N1 medium (soybean powder 3 g L-1, yeast extract 3 g L-1, trehalose 1 g L-1, L-proline 1 g L-1, beef extract 3 g L-1, glycerol 6 g L-1, K2HPO4 0.3 g L-1, MgSO4·7H2O 0.5 g L-1, FeSO4·7H2O 0.5 g L-1, CaCO3 2 g L-1, sea salt 30 g L-1, pH 7.2-7.4) in 250 mL flask by shaking at 200 rpm and 28 ºC for 5−7 days. 5 mL of fermentation broth was extracted with 5 mL butanone, and the extract was then dried under vacuum. The residue was dissolved into 70 μ L of CH3OH and subjected to HPLC analysis using a reversed-phase (Phenomenex Kinetex C18, 250 × 4.6 mm, 5 μ m) with UV detection at 265 nm on Agilent 1360 Infinity series workstation, the program was set as follows: solvent system (solvent A, 10% acetonitrile in water supplementing with 0.1% formic acid; solvent B, 90% acetonitrile in water); 5% B to 100% B (0−20 min), 100% B (20−30 min), 100% B to 5% B (30−32 min), 5% B (32−35 min), flow rate at 1 mL min-1.
Fermentation, Extraction, and isolation
The scaled up fermentation was carried out using spores directly in 250 mL flask. Briefly, the spores of Streptomyces sp. SCSIO 01127/∆lobP1 was inoculated into a 250 mL flask containing 50 mL of N1 medium and grown at 28 ºC for 6 days at 200 rpm. Subsequently, the fermentation cultures were centrifuged (3900 rpm, 15 min) to yield the supernatant and mycelia cake. A total of 12 L culture was prepared for isolating intermediates from Streptomyces sp. SCSIO 01127/∆lobP1 . The metabolites in supernatants were absorbed by XAD-16 resins, and the collected resins were eluted with 6 L acetone. The mycelia cake was extracted four times with 4 L acetone. After evaporation of organic solvents under vacuum, the residues were combined and re-extracted each time with 1 L butanone for eight rounds. After drying under vacuum, the extract (8 g) was then subjected to normal phase silica gel column (100−200 mesh) and eluted with a gradient of CHCl3/CH3OH (1:0, 4:1, 2:1, 0:1, v/v, 600 mL) to yield 4 fractions (Fr.1 to Fr.4). Fr.2 was further separated by Sephadex LH-20, eluted with CHCl3/CH3OH (1:1, v/v) to obtain 4 sub-fractions (Fr.2.1 to Fr.2.4). Fr.2.2 was purified by MPLC with ODS column, eluted with a linear gradient under the following program: solvent system (solvent A, water supplementing with 0.1% formic acid; solvent B, acetonitrile); 0% B to 60 % B (0−40 min), 60% B to 85% B (40−80 min), 85% B to 100% B (80−100 min), 100% B (100−120 min), flow rate at 20 mL min-1 to yield 9 sub-fractions (Fr. 2.2.1 to Fr. 2.2.9). Fr. 2.2.2 was purified by semi-preparative HPLC to yield 3 (5 mg), 4 (4.5 mg), 5 (7.6 mg),7 (8.1 mg), 8 (11.6 mg); Fr. 2.2.8 was purified by semi-preparative HPLC to afford 6 (305 mg).
Lobophorin N1 (3 ): white powder; [α ]25 D‒79.50 (c 0.08, CH3OH); UV (CH3OH)λ max (log ε ) 205 nm (4.36), 237 nm (3.83), 265 nm (3.72); 1H NMR (700 MHz, DMSO-d 6) and 13C NMR (175 MHz, DMSO-d 6) data: see Table S3; IR (film)ν max 3360, 2922, 1730, 1631, 1543, 1060, 1012, 866 cm-1; HRESIMS m/z 1155.5854 [M - H]- (calcd for C60H88N2O20, 1155.5857).
Lobophorin N2 (4 ): white powder; [α ]25 D‒87.25 (c 0.28, CH3OH); UV (CH3OH)λ max (log ε ) 205 nm (4.31), 235 nm (3.87), 266 nm (3.76); 1H NMR (700 MHz, DMSO-d 6) and 13C NMR (175 MHz, DMSO-d 6) data: see Table S3; IR (film)ν max 3450, 2931, 1732, 1635, 1541, 1058, 995, 866 cm-1; HRESIMS m/z 1140.6117 [M - H]- (calcd for C61H91NO19, 1140.6112).
Lobophorin N3 (5 ): white powder; [α ]25 D‒88.48 (c 0.13, CH3OH); UV (CH3OH)λ max (log ε ) 205 nm (4.37), 235 nm (3.83), 265 nm (3.68); 1H NMR (700 MHz, DMSO-d 6) and 13C NMR (175 MHz, DMSO-d 6) data: see Table S3; IR (film)ν max 2924, 2358, 1732, 1633, 1541, 1417, 1060, 1008, 866 cm-1; HRESIMS m/z 1197.6325 [M - H]- (calcd for C63H94N2O20, 1197.6327).
Overexpression and purification of LobP1, Fdx and FdR
The lobP1 gene was PCR amplified from the genomic DNA ofStreptomyces sp. SCSIO 01127 using primers LobP1EF/ER (Table S2). PCR products were assembled with the linearized pET28a (digested byNde Ⅰ/Bam HⅠ) using Single One Step Clone Kit (Vazyme Biotech) to afford pCSG5662 (Table S1) after sequencing confirmation. Expression of lobP1 were carried out in E. coli Rosetta (DE3)/pCSG5662 following standard procedures. The purification of (His)6-tagged LobP1 was performed by Ni2+-NTA affinity chromatography (GE Healthcare) with standard protocols. The purified and desalted LobP1 proteins were stored in storage buffer (50 mM HEPES buffer, 100 mM NaCl, 1 mM DTT, 10% glycerol, pH 7.6) at -80 °C until use. To obtain redox partners ferrodoxin (Fdx) and ferrodoxin reductase (FdR) from cyanobacteriumSynechococcus elongatus PCC7942,[24] the two expression plasmids pET28b-fdx_1499 and pET28b-fdR_0978 (Table S1) were transformed into E. coli BL21 (DE3), respectively. The expression and purification of Fdx and FdR were carried out in the same way as that of LobP1.
Enzyme assays of LobP1
To assay the enzymatic activities of LobP1 towards various substrates, each 100 μ L of reaction mixtures were prepared to contain the corresponding LOB substrate (400 μ M), LobP1 (1.8 μ M), Fdx/FdR (each 5 μ M) and NADPH (2 mM), in PBS buffer (50 mM, pH 7.6). Reactions were performed at 30 °C for 1 h. The reactions were quenched by adding 100 μ L methanol. The enzyme assays were monitored via HPLC analysis under the condition described above. To determine the kinetic parameters of LobP1 toward LOB N1 (3 ), the enzyme assay contains LobP1 (1.25 nM), saturating Fdx/FdR (each 5μ M), NADPH (1 mM) and 3 (2.5, 5, 10, 20, 50, 75, 100μ M) in PBS buffer (50 mM, pH 7.6); for the substrate LOB E (6 ), the enzyme assay contains LobP1 (2.5 nM), saturating Fdx/FdR (5 μ M each), NADPH (1 mM) and 6 (1, 2.5, 5, 10, 20, 50, 100 μ M), in PBS buffer (50 mM, pH 7.6); for the substrate LOB N (7 ), the enzyme assay contains LobP1 (12.5 nM), saturating Fdx/FdR (each 5 μ M), NADPH (1 mM) and 7 (5, 10, 20, 40, 50, 100, 150, 200, 400 μ M), in PBS buffer (50 mM, pH 7.6);. Each reaction mixture (100 μ L) was performed in triplicates and was incubated at 30 °C for 5 min, quenched by adding 100μ L methanol. Kinetic parameters were calculated by nonlinear regression analysis using GraphPad Prism 6 software.
Antibacterial assays
Antimicrobial activities were measured against four indicator strains,Bacillus subtilis 1064, Micrococcus luteus SCSIO ML01,Staphylococcus aureus ATCC 29213 and MRSA shhs-A1, by the broth microdilution method. The methods for the culturing, diluting, sampling and recording were the same as previously described.[13]