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DOI 10.1007/s00449-013-1007-2
ORIGINAL PAPER
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HPLC analysis of daptomycin externally using a mixture of five peptide standards. Accel-
erating voltages applied for MS and MS/MS measurements
Concentrations of daptomycin in reaction samples were were 20 and 8 kV, respectively. In MS/MS mode, collision
performed as described previously with modification [18]. In energy of 1 kV was applied and nitrogen was used as a
brief, samples were measured by HPLC method (1200 Ser- collision gas in collision-induced dissociation experiments.
ies, Agilent Company, USA) with Sunfire-C18 reversed- Raw spectral data were further processed using DataEx-
phase column (4.6 9 250 mm, 5 lm, ZORBAX SB-C18). plorer 4.6 software (Applied Biosystems).
A mobile phase of 0.1 % (v/v) trifluoroacetic acid in water
and acetonitrile (55:45, v/v) was used at a flow rate of Scanning electron microscopy (SEM)
1.0 mL/min. The detection wavelength and temperature was
at 218 nm and 30 °C, respectively. Peak areas for dapto- Samples from S. roseosporus were cultivated in optimal
mycin showed linear correlation with standard curves within medium at 30 °C, 200 rpm in batch fermentation for 24 and
the range of 0–200 mg/L. The standard deviation of three 192 h in terms of biomass of 15.26 and 8.27 g/L, respec-
repeated injections was normally below 3 %. tively. Then, 10 ml broth was centrifuged at 8,000 rpm for
10 min. The cell pellets were washed with deionized water
SDS-PAGE and tandem MS/MS analysis twice and the supernatant was removed. The rest cells for
SEM were prepared by placing a small drop of cell re-sus-
SDS-PAGE analysis was carried out on intracellular proteins pension on a formvar-coated cover glass. Excess solution
from cells that were crushed by homogenizer at 30 kpsi was wiped away using a piece of filter paper. Samples were
(Constant system, One Shot Model, UK) for one cycle, and fixed for 2 h by the addition of 2.5 % (v/v) glutaraldehyde
samples were collected by centrifugation at 12,0009g for and then dehydrated using a graded ethanol series and 100 %
15 min. The gel was prepared with 0.1 % (w/v) SDS in 10 % tert-butyl alcohol. All samples were fixed, embedded, and
(w/v) separating gel (containing 10 % acrylamide) and 4 % sectioned under anaerobic conditions to avoid oxidation of
(w/v) stacking gel (containing 4 % acrylamide). Tris–gly- redox-sensitive components. Whole mounts were examined
cine buffer (pH 8.3) containing 0.1 % (w/v) SDS was used as using a Hitachi S-4800 SEM instrument (Tokyo, Japan)
the electrode buffer. Samples were treated with the sample operating at a 20 kV accelerating voltage.
buffer and heated at 100 °C for 5 min prior to application to
the gel. Electrophoresis was run from the cathode to anode at
a constant current of 20 mA per slab at room temperature in a Results and discussion
Bio-rad mini gel electrophoresis unit, and proteins were
visualized by staining with Coomassie blue R-250. The Effect of precursors on daptomycin production
candidates of protein then were digested by trypsin [19] and
carried out by MALDI-TOF–MS/MS analysis in positive As indicated in Table 2, decanoic acid and sodium decanoate
ionization mode using the Applied Biosystems, SCIEX TOF/ as the precursors were compared in the optimization of
TOFTM, 5,800 System (Applied Biosystems, MA, USA). daptomycin production. Almost no daptomycin is produced
The instrument was equipped with a solid-state laser (diode from S. roseosporus without decanoic derivative chemicals
pumped Nd: YAG laser) pulsing at a repetition rate of 1 kHz. addition. However, the decanoic acid (a type of organic acid)
A solution of 50 % (v/v) acetonitrile and 0.1 % (v/v) TFA in might be a kind of the toxic element in culture of
methanol was used as a MALDI matrix. Both MS and MS/ S. roseosporus [20], in terms of the observation (or fact) that
MS spectra were acquired using dual-stage reflectron mirror the biomass dropped down from 25.34 to 10.35 g/L. Besides,
and accumulated up to 2,500 and 5,000 shots in MS and MS/ the production of daptomycin was only 0.81 mg/L when
MS mode, respectively. The instrument was calibrated decanoic acid was used. By contrast, biomass, daptomycin
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Table 2 Precursors effect on the biomass and daptomycin production of Streptomyces roseosporus cultured in the flask at 30 °C, 200 rpm for
240 h
Precursors Biomass (g/L) DAPa Specific DAP Relative
production (mg/L) production (mg/g DCWb) production ratioc
None 25.34 nd nd nd
Decanoic acid 10.35 0.81 0.078 1.0
Sodium decanoate 26.78 50.65 2.153 71.55
a
DAP is the abbreviation of daptomycin
b
DCW is the abbreviation of dry cell weight
c
Relative production ratio was the ratio of DAP production (mg/L) and biomass (g/L)
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Table 3 Mascot protein identification of major intracellular proteins differed from precursor effect on Streptomyces roseosporus at time series
Code Protein Strain species Protein score Accession no. Protein Mw (Da)
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1 50 0 0 8 24.4 23.2
2 50 5 0.3 10 28.2 20.4
3 50 10 0.7 12 35.3 27.3
4 50 15 1.0 14 38.5 25.6
5 60 0 0.3 12 38.7 24.5
6 60 5 0 14 29.4 18.0
7 60 10 1.0 8 57.0 28.3
8 60 15 0.7 10 56.3 28.9
9 70 0 0.7 14 46.1 26.8
10 70 5 1.0 12 49.8 27.5
11 70 10 0 10 37.6 24.1
12 70 15 0.3 8 36.9 23.2
13 80 0 1.0 10 29.2 17.5
14 80 5 0.7 8 25.3 16.6
15 80 10 0.3 14 30.1 17.3
16 80 15 0 12 12.5 12.1
DAP production
K1e 126.4 138.4 103.9 143.6
K2 181.4 132.7 133.9 151.3
K3 170.4 160.0 163.0 136.3
Fig. 4 Optimization of carbon source (a) and nitrogen source (b). K4 97.1 144.2 174.5 144.1
Cultivation of streptomyces roseosporus was performed at 30 °C, Rf 84.3 27.3 70.6 15.0
200 rpm. Biomass (black); daptomycin concentration (red); specific Biomass production
productivity (green). DextrinM-: absence of molasses in original
medium (color figure online) K1 96.5 92 77.4 91.3
K2 99.7 82.5 85.4 86.4
K3 101.6 97 99.6 91.4
K4 63.5 89.8 94.4 87.7
Yeast extract has been considered an ideal nitrogen source R 38.1 14.5 22.2 5.0
for many secondary metabolites by Streptomyces micro- a
A: major carbon: dextrin
organism [27–29]. b
B: second carbon: glucose
By following an orthogonal experimental design (L16) c
C: additional carbon: molasses
(Table 1), the major carbon, dextrin, was found to be the d
key factor contributing to the overall yield of biomass and D: major nitrogen: yeast extract
e
daptomycin production. R values revealed that the impact K value is the sum of daptomycin productions in a certain level of a
factor
of medium compositions on daptomycin production and f
R value is the maximum K value of a factor minus the minimum
biomass were in the order of dextrin, molasses, glucose, one
and yeast extract (Table 4). The best yield of biomass and
daptomycin were 28.3 and 57.0 g/L in run 7. Herein, the
optimal concentrations of medium, with respect to minimal carbon source. Thus, fed-batch strategy was based on the
composition and high daptomycin production, were deter- flow rate of dextrin as a feedback control. From the results
mined to be 60 g/L of dextrin, 10 g/L of glucose, 1.0 g/L in Fig. 5a, similar cell growth behaviors were demon-
of molasses, and 8.0 g/L of yeast extract. strated in flask, batch, and fed-batch fermentation. How-
ever, the production of daptomycin was effectively
Improved daptomycin production by fed-batch strategy increased in the fed-batch strategy. It reached the highest
productivity of daptomycin to 812.0 mg/L (Fig. 5b).
For the aim of industrial application, an improved fed- Otherwise, the daptomcyin concentrations were 71.9 mg/L
batch strategy was established. According to the previous in flask and 217.5 mg/L in batch fermentation, respec-
results, dextrin was the most critical nutrient and major tively. Contrary to the original fermentation in flask, the
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