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]