Bioprocess Biosyst Eng
DOI 10.1007/s00449-013-1007-2
ORIGINAL PAPER
Daptomycin antibiotic production processes in fed-batch
fermentation by Streptomyces roseosporus NRRL11379
with precursor effect and medium optimization
I-Son Ng • Chiming Ye • Zhixiang Zhang •
Yinghua Lu • Keju Jing
Received: 18 April 2013 / Accepted: 23 June 2013
Ó Springer-Verlag Berlin Heidelberg 2013
Abstract Sodium decanoate was first found to be an
effective precursor for synthesis of daptomycin from
Streptomyces roseosporus NRRL11379 which was
increased to 71.55-fold, compared with decanoic acid. The
optimal flow rate of precursor was at 600 mg/(L day) after
48 h fermentation. From protein analysis via SDS-PAGE
and identification of Tandem MS/MS afterwards, it deci-
phered that guanosine pentaphosphate synthetase, PNPase,
tripeptidylamino peptidase primarily dealing with dapto-
mycin synthesis. By applying Taguchi’s L16 in culture
optimization, the best yield was obtained from the medium
with 60 g/L dextrin, 10 g/L dextrose, 1.0 g/L molasses,
and 8 g/L yeast extract, respectively. The fed-batch fer-
mentation, applied with feedback control of dextrin, stim-
ulated the production up to 812 mg/L at 288 h. To our best
knowledge, the daptomycin production in this study is
significantly higher than that in previous studies and can
make it more widely used in pharmaceutical industry.
Keywords Daptomycin antibiotic
Streptomyces
roseosporus
Fed-batch fermentation
Optimization

Sodium decanoate
C. Ye and Z. Zhang contributed equally to this work.
I.-S. Ng (&)
C. Ye
Z. Zhang
Y. Lu
K. Jing
Department of Chemical and Biochemical Engineering,
College of Chemistry and Chemical Engineering,
Xiamen University, Xiamen 361005, China
e-mail: yswu@xmu.edu.cn
I.-S. Ng
Y. Lu
K. Jing
The Key Laboratory for Synthetic Biotechnology
of Xiamen City, Xiamen University, Xiamen 361005, China
Introduction
Secondary metabolites produced by microorganisms are
the sources of many drugs, including antibiotics, antitumor
compounds, immune-suppressants, antiviral and antipara-
sitic agents, and enzyme-inactivating compounds [1]. They
offer a promising source for new bioactive compounds
synthesis due to its structural diversity, particularly those
produced by the large multi-modular non-ribosomal pep-
tide synthetases (NRPSs) and polyketide synthases (PKS)
[2]. However, the production of these secondary metabo-
lites is always poor in most instances. To improve the
production of metabolites, various disciplines involved in
the research and some achievements have been obtained
[3–5].
Antibiotic, A21978C was first isolated from Strepto-
myces roseosporus in 1980 [6]. The A21978C is a member
of A21978 family, while other A21978 factors (A21978A,
-B, -D, -E) were accompanying products. As an analog of
A21978C, daptomycin (C72H101N17O26, Mw 1,620.67) is
an important clinical therapeutic agent which is derived
from the fermentation of S. roseosporus [7, 8]. It is
approved to be effective for treatment of skin and skin
structure infections caused by gram-positive pathogens [9].
Its mode of action is distinct from any other approved
antibiotic as it rapidly kills gram-positive bacteria by dis-
rupting multiple aspects of bacterial membrane function
[10, 11], which is calcium- and potassium-related [12, 13].
Production of daptomycin was first used in the fer-
mentation of S. roseosporus [10, 14]. Nevertheless, the
production of daptomycin was still insufficient and unsta-
ble as the product was toxic and inhibited the microor-
ganism itself. Various strategies, such as medium
optimization, fermentative strategy, genetic engineering
modification, and metabolic flux analysis have been
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established to achieve the overproduction of daptomycin
[4, 15, 16] although previous reports note that S. roseosp-
orus NRRL collection has been mutagenic and enhanced
the daptomycin production via effect of cofactors in the
fermentation [15, 16]. To the best of our knowledge, there
are still no reports describing the effect of precursor,
sodium decanoate, and minimizing medium composition to
improve the yield of daptomycin. Attempts were made to
explore the precursor effects with protein analysis via SDS-
PAGE and identification of Tandem MS/MS, optimal car-
bon source and nitrogen source, and the optimization of
medium composition using Taguchi’s orthogonal arrays
(L16). Fed-batch fermentation strategy followed by mor-
phologies of cells via scanning electron microscopy (SEM)
analysis for examination of S. roseosporus was also
investigated.
Materials and methods
Microorganism and medium
Streptomyces roseosporus NRRL11379 was purchased
from NRRL collection. The microorganism was grown on
YMD agar plate at 30 °C and maintained at 4 °C for long
storage. The YMD agar plate (g/L) contained dextrose 4,
malt extract 10, yeast extract 4, CaCO3 2, agar 15. The
composition of the primary and secondary seed medium
(g/L) was trypticase soy broth 30, dextrin 25. The tertiary
seed medium (g/L) contained soy bean flour 28.8,
Fe(NH4)2SO4
6H2O 0.9, dextrose 17, potato dextrin 71.9,
cane molasses 7.2. Fermentation medium (g/L) contained
yeast 11, Fe(NH4)2SO4
6H2O 0.86, dextrose 10.7, potato
dextrin 72, cane molasses 7.2. A 50 ll of 300 mg/L biotin
and 100 ll of 300 mg/L lipoic acid was added to the tertiary
seed medium and fermentation medium before inoculation,
respectively. All materials were autoclaved at 121 °C for
20 min and afterwards the pH was adjusted to 7.0.
Culture of S. roseosporus
A disc of YMD with a diameter of 0.4 cm containing
approximately 105–106 spores was inoculated into the
250-mL flask containing 50 mL primary seed medium and
incubated at 30 °C, 200 rpm for 48 h. The primary seed
medium was inoculated with 5 % (v/v) into fresh 50 mL
secondary seed medium and cultured at same condition for
24 h. A 5 % (v/v) inoculation volume of the secondary
seed medium was inoculated into another 50 mL tertiary
seed medium and cultured for another 24 h. Then the ter-
tiary seed medium was inoculated into the fermentation
medium. After that, biomass (dry cell weight) and dapto-
mycin concentration were analyzed daily.
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Bioprocess Biosyst Eng
Optimization of precursor addition
Decanoic acid and sodium decanoate with a final concen-
tration of 0.02 % (w/v) in the fermentation medium were
be selected as the precursor. They were added every 12 h
after the second day of fermentation and till the end of
culture. For the optimal flow rate of precursor, sodium
decanoate in terms of mg/(L day) was chosen as 400, 500,
600, 700, 800, and 900 in fermentation, respectively.
Subsequently, addition time was optimized between 0 and
5 days. All experiments were performed in duplicate.
Optimal of carbon and nitrogen sources
For obtaining the optimum carbon and nitrogen sources,
five carbon sources: dextrin, glucose, sucrose, starch, and
dextrinM- (absence of molasses in original medium) of
60 g/L and eight nitrogen sources: NaNO3, NH4Cl, malt
extract, tryptone, yeast extract, soybean powder, soybean
peptone, and beef extract of 10 g/L were studied through
one-factor-at-a-time method. All experiments were per-
formed in duplicate.
Orthogonal experimental design (L16)
To improve the production of daptomycin, orthogonal
experimental design was performed in the study. Several
factors such as major carbon source dextrin, second carbon
source glucose, additional carbon source molasses, and
major nitrogen source yeast extract were determined by the
orthogonal experiments of Taguchi’s L16 in Table 1, for
which four levels of four factors were designed.
Fed-batch fermentation in bioreactor
Fermentation was performed at 30 °C in a 3.6-L biore-
actor (Applikon, Holland) with an initial culture volume
of 1.5 L. Inoculation was 5 % (v/v) and the aeration rate
was controlled at 3.5 vvm. Dissolved oxygen was
maintained above 30 % of the saturated dissolved oxy-
gen concentration by automatically adjusting the agita-
tion speed above 450 rpm. Aqueous solution containing
2 % (w/v) sodium decanoate precursor was added at the
rate of 35 lL/min after 24 h in supplement. For the fed-
batch fermentation, the dextrin was added when the
carbon source had been consumed below 20 g/L of
reducing sugar content while at the same time the flow
rate of precursor was changed to 3.5 lL/min. During the
process of fermentation, biomass was determined by dry
cell weight from 80 °C for 24 h, pH value was main-
tained at 6.5 with 1 N NH4OH, daptomycin concentra-
tion was analyzed by HPLC and residual sugar content
was measured by DNS method [17].
Bioprocess Biosyst Eng
Table 1 Four levels of four
Levels and Major carbon
factors in the orthogonal
factors dextrin (g/L)
experimental design (L16) of
Streptomyces roseosporus 1 50
fermentation
2 60
3 70
4 80
HPLC analysis of daptomycin
Concentrations of daptomycin in reaction samples were
performed as described previously with modification [18]. In
brief, samples were measured by HPLC method (1200 Ser-
ies, Agilent Company, USA) with Sunfire-C18 reversed-
phase column (4.6 9 250 mm, 5 lm, ZORBAX SB-C18).
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
1.0 mL/min. The detection wavelength and temperature was
at 218 nm and 30 °C, respectively. Peak areas for dapto-
mycin showed linear correlation with standard curves within
the range of 0–200 mg/L. The standard deviation of three
repeated injections was normally below 3 %.
SDS-PAGE and tandem MS/MS analysis
SDS-PAGE analysis was carried out on intracellular proteins
from cells that were crushed by homogenizer at 30 kpsi
(Constant system, One Shot Model, UK) for one cycle, and
samples were collected by centrifugation at 12,0009g for
15 min. The gel was prepared with 0.1 % (w/v) SDS in 10 %
(w/v) separating gel (containing 10 % acrylamide) and 4 %
(w/v) stacking gel (containing 4 % acrylamide). Tris–gly-
cine buffer (pH 8.3) containing 0.1 % (w/v) SDS was used as
the electrode buffer. Samples were treated with the sample
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
Bio-rad mini gel electrophoresis unit, and proteins were
visualized by staining with Coomassie blue R-250. The
candidates of protein then were digested by trypsin [19] and
carried out by MALDI-TOF–MS/MS analysis in positive
ionization mode using the Applied Biosystems, SCIEX TOF/
TOFTM, 5,800 System (Applied Biosystems, MA, USA).
The instrument was equipped with a solid-state laser (diode
pumped Nd: YAG laser) pulsing at a repetition rate of 1 kHz.
A solution of 50 % (v/v) acetonitrile and 0.1 % (v/v) TFA in
methanol was used as a MALDI matrix. Both MS and MS/
MS spectra were acquired using dual-stage reflectron mirror
and accumulated up to 2,500 and 5,000 shots in MS and MS/
MS mode, respectively. The instrument was calibrated
Second carbon Additional carbon Major nitrogen yeast
glucose (g/L) molasses (g/L) extract (g/L)
0 0 8
5 0.3 10
10 0.7 12
15 1.0 14
externally using a mixture of five peptide standards. Accel-
erating voltages applied for MS and MS/MS measurements
were 20 and 8 kV, respectively. In MS/MS mode, collision
energy of 1 kV was applied and nitrogen was used as a
collision gas in collision-induced dissociation experiments.
Raw spectral data were further processed using DataEx-
plorer 4.6 software (Applied Biosystems).
Scanning electron microscopy (SEM)
Samples from S. roseosporus were cultivated in optimal
medium at 30 °C, 200 rpm in batch fermentation for 24 and
192 h in terms of biomass of 15.26 and 8.27 g/L, respec-
tively. Then, 10 ml broth was centrifuged at 8,000 rpm for
10 min. The cell pellets were washed with deionized water
twice and the supernatant was removed. The rest cells for
SEM were prepared by placing a small drop of cell re-sus-
pension on a formvar-coated cover glass. Excess solution
was wiped away using a piece of filter paper. Samples were
fixed for 2 h by the addition of 2.5 % (v/v) glutaraldehyde
and then dehydrated using a graded ethanol series and 100 %
tert-butyl alcohol. All samples were fixed, embedded, and
sectioned under anaerobic conditions to avoid oxidation of
redox-sensitive components. Whole mounts were examined
using a Hitachi S-4800 SEM instrument (Tokyo, Japan)
operating at a 20 kV accelerating voltage.
Results and discussion
Effect of precursors on daptomycin production
As indicated in Table 2, decanoic acid and sodium decanoate
as the precursors were compared in the optimization of
daptomycin production. Almost no daptomycin is produced
from S. roseosporus without decanoic derivative chemicals
addition. However, the decanoic acid (a type of organic acid)
might be a kind of the toxic element in culture of
S. roseosporus [20], in terms of the observation (or fact) that
the biomass dropped down from 25.34 to 10.35 g/L. Besides,
the production of daptomycin was only 0.81 mg/L when
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)

the first attempt to discover sodium decanoate as an effective

Fig. 1 Optimal concentration and flow rate of precursor sodium
decanoate. Cultivation of Streptomyces roseosporus was performed at
30 °C, 200 rpm. Biomass (filled square); daptomycin concentration
(filled triangle), and specific productivity (filled circle)
Fig. 2 Optimal for addition time of precursor sodium decanoate.
Cultivation of Streptomyces roseosporus was performed at 30 °C,
200 rpm. Biomass (black); daptomycin concentration (red); specific
productivity (green) (color figure online)
precursor for daptomycin by S. roseosporus.
From this point of view, further optimization of the
addition amount and addition time of sodium decanoate
was carried out. As shown in Fig. 1, specific daptomycin
production increased along with addition of sodium de-
canoate from 400 to 900 mg/(L day). The optimal addition
was presented at 600 mg/(L day) of sodium decanoate for
the largest amount of daptomycin produced and biomass
was as well kept at 26.7 g/L. The high titer of precursor
would be poisonous to the cell as the biomass decreased
sharply after 800 mg/(L day). Figure 2 revealed that the
addition time of precursor with maximum daptomycin
production was achieved when sodium decanoate was
added at the second day after inoculation. Delaying the
addition time of the precursor also prevented inhibition of
cell growth. Nevertheless, daptomycin production was not
stimulated after the second day.
Since sodium decanoate is a water-soluble substance [21]
it can promise a relatively high concentration and be absor-
bed by cells easily. As the precursor, sodium decanoate was
in favor of the daptomycin production compared with the
aliphatic fatty acids which were examined for enhancement
of other lipopeptide antibiotics [22]. However, as an addi-
tive, it also showed an inhibition on growth and metabolite
synthesis, when the concentration was too high. In conse-
quence, the addition time of the precursor was very critical. It
showed that late exponential phase was the best additive time
in Fig. 2. It is supposed that the enhanced level of interme-
diates in the antibiotic biosynthesis as well as up-regulated
expression of key biosynthetic genes mostly depended on a
critical time [23]; thus an addition sodium decanoate of
600 mg/(L day) at the second day after inoculation was
suggested as the optimal condition.

production and specific daptomycin production significantly Protein analysis

increased when sodium decanoate was used as the precur-
sor to achieve biomass of 26.78 g/L and maximum dapto- Further analysis in Fig. 3 for SDS-PAGE profiles of S. ros-
mycin production of 50.65 mg/L (Table 2). Actually, it was eosporus at 144, 96, and 48 h without and with sodium

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accurate function was still unknown as information is not

Fig. 3 SDS-PAGE profiles of intracellular proteins obtained from
cells of Streptomyces roseosporus at 144, 96 and 48 h without
precursor (lanes 2, 4, 6) and with sodium decanoate (lanes 3, 5, 7),
respectively. The proteins were extracted and subjected to 10 % (w/v)
SDS-PAGE followed by Coomassie blue R-250 staining. M (lane 1)
means protein markers in kDa is 5 lL of Fermentas prestained protein
ladderTM. The proteins which indicated by the arrows in the boxes
were followed by MALDI-TOF–MS/MS
decanoate indicated that the major different protein bands
were at ca. 130, 82, 72, 55, and 40 kDa, respectively. It is
easy to find that the differences of protein expression
between precursor and non-precursor group only displayed
at 96 and 144 h, but no difference at 48 h. The above dif-
ference occurred after precursor addition implied that the
proteins synthesis may be related to daptomycin synthesis.
To identify the major bands, they were subjected to in-
gel trypsin digestion followed by MALDI-TOF–MS/MS
analysis. Detailed mascot identification information
(Table 3) indicated that the mass spectrum identified the
proteins as the putative regulatory protein, guanosine
pentaphosphate synthetase, polyribonucleotide nucleotid-
yltransferase phosphorylase (PNPase), putative secreted
tripeptidylamino peptidase, and putative two-component
system histidine kinase. Among them, putative regulatory
protein is a kind of regulatory factor of enzymes, but the
enough. Guanosine pentaphosphate synthetase and polyri-
bonucleotide nucleotidyltransferase phosphorylase
(PNPase) are involved in purine metabolism (information
from KEGG website:
http://www.genome.jp/kegg-bin/show_pathway?ec00230)
which is closely related to amino acid metabolism. As
daptomycin consists of 13 amino acids, the two proteins
affect the amino acid metabolism of daptomycin synthesis.
For putative secreted tripeptidylamino peptidase, it con-
tains the serine active site thus to control the serine
metabolism in the 13 amino acids of daptomycin. Putative
two-component system histidine kinase is a signal trans-
duction protein and contains possible hydrophobic mem-
brane spanning regions in Streptomyces coelicolor [24],
and is supposed to regulate the secretion of daptomycin.
Further analysis of the mechanism of daptomycin synthesis
through transcription level expression, two-dimensional
electrophoresis, and western blotting are necessary.
Medium optimization
The effect of different carbon and nitrogen sources on
daptomycin production was shown in Fig 4a, b, respec-
tively. The dextrin, glucose, sucrose, starch, and dextrinM-
were considered in the comparison of carbon source. As
shown in Fig. 4a, the highest biomass and daptomycin
production were achieved when dextrin was used as the
major carbon source. As dextrin belongs to the polysac-
charides, it is supposed to be a sequential biodegradation
carbon source in cell culture [25]. Both sucrose and starch
were suitable in culture of S. roseosporus but lacked
stimulation for daptomycin. Besides, absence of molasses
also altered the production of daptomycin, implying the
importance of molasses. Almost all organic nitrogen
sources were acceptable for daptomycin production in S.
roseosporus, except for malt extract and inorganic nitrogen
sources (Fig. 4b). The nitrogen effect to S. roseosporus was
similar to S. hygroscopicus which the gene transcription
related to the nitrogen source in an antibiotic fermentation
[26]. Among of various nitrogen sources, yeast extract was
the best as it led to 53.4 mg/L in yield of daptomycin.

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)

A Putative regulatory protein Streptomyces coelicolor A3(2) 22 gi|5457283 122,280.0

B Guanosine pentaphosphate synthetase Streptomyces coelicolor A3(2) 67 gi|24413893 79,359.9
C Polyribonucleotide nucleotidyltransferase Streptomyces coelicolor A3(2) 86 gi|81445833 79,359.9
phosphorylase (PNPase)
D Putative secreted tripeptidylamino peptidase Streptomyces coelicolor A3(2) 136 gi|9716116 58,535.7
E Putative two-component system histidine kinase Streptomyces coelicolor A3(2) 24 gi|7672245 45481.9

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Table 4 Daptomycin production and biomass of Streptomyces ros-

eosporus in an orthogonal L16 experiments
Run Aa Bb Cc Dd Production Biomass
(g/L) (g/L) (g/L) (g/L) (DAP mg/L) (g/L)

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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in fermentation is often applicable in the improvement of

microbial natural products as well as on industrial scale
[30, 31]. The stimulation by fed-batch was generally
caused by the sufficient nutrients for secondary metabolites
or antibiotics. Sequentially, daptomycin production via
different strains was compared in Table 5. In this study,
daptomycin production showed a relatively high value than
other researches [16, 32, 33]. But the disadvantage was that
the cultivation cost took a very long time. Nevertheless, the
results reported here will be potent and certainly applicable
in industrial applications.

Morphology analysis of Streptomyces roseosporus

The morphological changes of cells during cultivation were

Fig. 5 Culture conditions comparison in 250 mL-flask (black), 3.6L-
batch (white) and 3.6L-fed batch (blue). a Biomass in circle symbols
and pH in square symbols, b daptomycin concentration and c specific
productivity of Streptomyces roseosporus at 30 °C, 200 rpm (color
figure online)
fed-batch fermentation was increased to 3.03- and 11.29-
fold compared with batch and flask culture. Finally, the
specific productivity of daptomycin was obtained for
144.4 mg/g-DCW (Fig. 5C). Fed-batch strategy developed
analyzed by SEM (Fig. 6). From the two different phases,
i.e. before the addition of precursor at 24 h and culture at
192 h with precursor addition, morphological elucidation
by SEM showed the difference in length and density of
cells. It was observed that mycelium of cells was long and
thick at 24 h in terms of biomass of 15.26 g/L without
daptomycin produced, and changed to a short, thin, and
apical form after production of daptomycin at 192 h in
terms of biomass of 8.27 g/L with 168.12 mg/L of dapto-
mycin produced. It was concluded that the addition of
precursor enhances the accumulation of daptomycin, which
inhibits the cell growth and leads to the structure disruption
of mycelium. This result was consistent with the steep drop
of biomass after stationary phase.
Scanning electron microscopy and atomic force
microscopy had been adopted in the analysis of fungal cells
[34]. The obvious fine hair-like structure existed on the
spores and hyphae from Streptomyces species [35] while
morphological compartments of S. olindensis became api-
cal, subapical, and hyphal in production of antibiotic, re-
tamycin [36]. We suggested that SEM offline monitor
would be an alternative approach to elucidate antibiotic
production at optimal growth phase.

Table 5 Comparison of daptomycin production in various strains

Strains Daptomycin or A21978Ca (mg/L) Working volume (L) Culture time (h) References

Streptomyces roseosporus LC-51 632 7.5 132 Yu et al. [16]

Streptomyces roseosporus NRRL11379 296 (A21978C) 7.0 144 Lu et al. [32]
Streptomyces lividans TK23 and TK64 55 (A21978C) – 168–240 Penn et al. [33]
Streptomyces roseosporus NRRL11379 812 3.6 282 This study
a
A21978C is indicated to cyclic lipopeptide

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Fig. 6 SEM images of
S. roseosporus at different
culture time. Left: cultured for
24 h (-) which was without
daptomycin production; right:
cultured for 192 h (?) which
reached to almost the maximum
production
Conclusions
In the current study, daptomycin production via Strepto-
myces roseosporus NRRL11379 by optimization of pre-
cursor, medium compositions, and fermentation strategy
has first been reported. We successfully established a cost-
effective medium and feedback controlling approach, by
utilizing dextrin as the major carbon source in fed-batch
fermentation, to produce highly antibiotic daptomycin.
Furthermore, the protein analysis with sodium decanoate
added to S. roseosporus as precursor and hyphal changes
when daptomycin secretion increased were observed
through SDS-PAGE coupled with Tandem MS/MS and the
images from SEM, respectively.
Acknowledgments The authors are grateful to the financial support
by the Fundamental Research Funds for the Central Universities
(2011121017), the Chinese National Natural Science Foundation
(21206141) and the Fujian Provincial Department of Science and
Technology (2012I0009).
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