jmb

J. Microbiol. Biotechnol. (2014), 24(10), 1319–1326

http://dx.doi.org/10.4014/jmb.1403.03024 Research Article

Review

Production of Rapamycin in Streptomyces hygroscopicus from

Glycerol-Based Media Optimized by Systemic Methodology S
Yong Hyun Kim1, Bu Soo Park2, Shashi Kant Bhatia1, Hyung-Min Seo1, Jong-Min Jeon1, Hyun-Joong Kim1,
Da-Hye Yi1, Ju-Hee Lee1, Kwon-Young Choi3, Hyung-Yeon Park4, Yun-Gon Kim5, and Yung-Hun Yang1,6*
1
Department of Microbial Engineering, Konkuk University, Seoul 143-701, Republic of Korea
2
Samyang Biopharmaceuticals Corporation 730, Daejeon 305-717, Republic of Korea
3
Department of Environmental Engineering, Ajou University, Suwon 443-749, Republic of Korea
4
Bio-MAX Institute, Seoul National University, Seoul 151-742, Republic of Korea
5
Chemical Engineering, Soongsil University, Seoul 156-743, Republic of Korea
6
Institute for Ubiquitous Information Technology and Applications (CBRU), Konkuk University, Seoul 143-701, Republic of Korea

Received: March 10, 2014

Revised: May 29, 2014 Rapamycin, produced by the soil bacterium Streptomyces hygroscopicus, has the ability to
Accepted: July 3, 2014
suppress the immune system and is used as an antifungal, anti-inflammatory, antitumor, and
immunosuppressive agent. In an attempt to increase the productivity of rapamycin,
mutagenesis of wild-type Streptomyces hygroscopicus was performed using ultraviolet
First published online radiation, and the medium composition was optimized using glycerol (which is one of the
July 7, 2014 cheapest starting substrates) by applying Plackett-Burman design and response surface
*Corresponding author methodology. Plackett-Burman design was used to analyze 14 medium constituents: M100
Phone: +82-2-450-3936; (maltodextrin), glycerol, soybean meal, soytone, yeast extract, (NH4)2SO4, L-lysine, KH2PO4,
Fax: +82-2-3437-8360;
E-mail: [email protected]
K2HPO4, NaCl, FeSO4·7H2O, CaCO3, 2-(N-morpholino) ethanesulfonic acid, and the initial pH
level. Glycerol, soytone, yeast extract, and CaCO3 were analyzed to evaluate their effect on
rapamycin production. The individual and interaction effects of the four selected variables
were determined by Box-Behnken design, suggesting CaCO3, soytone, and yeast extract have
negative effects, but glycerol was a positive factor to determine rapamycin productivity.
Medium optimization using statistical design resulted in a 45% (220.7 ± 5.7 mg/l) increase in
rapamycin production for the Streptomyces hygroscopicus mutant, compared with the
S upplementary data for this unoptimized production medium (151.9 ± 22.6 mg/l), and nearly 588% compared with wild-
paper are available on-line only at
type Streptomyces hygroscopicus (37.5 ± 2.8 mg/l). The change in pH showed that CaCO3 is a
http://jmb.or.kr.
critical and negative factor for rapamycin production.
pISSN 1017-7825, eISSN 1738-8872

Copyright © 2014 by Keywords: Streptomyces hygroscopicus, immunosuppressant, rapamycin production, Plackett-

The Korean Society for Microbiology Burman design, Box-Behnken design, response surface methodology
and Biotechnology

Introduction [18]. It is also reported that rapamycin can extend the

lifespan of mice [3]. Thus, there has been great interest in
Rapamycin, produced by Streptomyces hygroscopicus, is a increasing the yield of rapamycin, and many researchers
31-membered macrocyclic natural product exhibiting have mainly focused on increasing rapamycin titers [1, 9,
various biological and pharmacological activities, including 22, 25] and the production of rapamycin analogs to enhance
antifungal, immunosuppressive, antitumor, neuroprotective, the efficacy of rapamycin [16]. A number of metabolic
and anti-aging activities [5, 23]. Its potent activity, unique engineering approaches have been tried so far; however,
mode of action, and low toxicity have led to a great deal of these efforts have not been successful as the titer is still too
interest in its potential applications in human medicine low to produce rapamycin efficiently and economically [8,

October 2014 ⎪ Vol. 24 ⎪ No. 10

1320 Kim et al.

18, 21]. As a result, mutagenesis is still a feasible solution to or Sigma-Aldrich (St. Louis, MO, USA). For the fermentation,
improve the bacterial strain for higher productivity and S. hygroscopicus was cultivated at 30°C in 50 ml [12] of production
ultaviolet (UV) radiation induces different evolutionary medium (30 g M100, 30 g glycerol, 10 g soybean meal, 10 g soytone,
changes on bacteria that would otherwise not be possible 6.5 g yeast extract, 5 g (NH4)2SO4, 6.5 g L-lysine, 0.7 g KH2PO4,
1.14 g K2HPO4, 5 g NaCl, 0.05 g FeSO4·7H2O, and 42.6 g MES, pH
by metabolic engineering. However, it still needs optimization
5.5, per liter) with constant shaking at 200 rpm. This medium is
because it is strongly influenced by media compositions
called the unoptimized medium in this experiment. For UV
[20].
random mutagenesis, we used UV-mutagenesis to improve the
One way to overcome or avoid this is to optimize the strain by the direct plate irradiation protocol [20], and the spore
rapamycin production conditions, and there is information suspension was diluted with sterile water and placed in an
on optimization of the rapamycin production using a small uncovered petri dish and irradiated under a UV lamp (kill rate
number of factors and examination of different carbon 99%) [26].
sources [6]. Although this information is very helpful to
design media composition, the effects from the different Assay for Rapamycin Production
medium components were not well evaluated for many For rapamycin extraction, after centrifugation of 500 µl of
cultured cells, the supernatant was discarded. Then 500 µl of
factors that may cause interactive effects on the final
methanol was added to the cells in a 1.7 ml Eppendorf tube, and
production.
the supernatant was analyzed by high-performance liquid
Glycerol is one of the cheapest starting materials as a
chromatography (HPLC, YL-9100, Korea) using a Waters C18
carbon source, and has been found to support the growth reverse phase column, acetonitrile-water (80:20 (v/v)) as the mobile
and antibiotic production [10]. Glycerol has attracted the phase, and 1 ml/min flow rate with detection at 277 nm [22].
attention of scientific and industrial communities owing to
its generation in bulk quantities as a byproduct of biofuel Statistical Analysis
industries. With the rapid growth of these industries in Minitab 16.0 (Minitab Inc., Pensylvania, USA) was used for the
recent years, glycerol is frequently treated as a very low- experimental designs and subsequent regressional analysis of the
value byproduct or even a waste product with a disposal experimental data. Statistical analysis of the model was
cost associated to it. Glycerol is not only abundant and performed to evaluate the analysis of variance (ANOVA). The
inexpensive but can also generate more reducing equivalents quality of the polynomial model equation was judged statistically
by the coefficient of determination R2, and its statistical
than glucose or xylose [2], and thus media optimization of
significance was determined by an F-test. The significance of the
glycerol-based media seems to be a very promising and
regression coefficients was tested by a t-test. In this case, glycerol,
economical route to produce rapamycin repeatedly and
soytone, CaCO3, and yeast extract all had a significant effect on
stably. To identify the effects and optimal medium for rapamycin yield (p < 0.05).
rapamycin production, information of other components
were gathered from previous reports and employed in the Screening of Essential Medium Components Using the Plackett–
Plackett-Burman design and response surface methodology Burman Design
for glycerol-based media. The Plackett-Burman design can The Plackett-Burman design was used to analyze important
help to find important factors by eliminating many factors. Twenty experiments were conducted in duplicate to
experiments and avoiding the drawbacks of one variable at evaluate 14 factors. A total of 14 components (independent
variables k = 14, Table 1) were selected for the study, with each
a time [14, 17, 18]. Dual application of the Plackett-Burman
variable being represented at two levels, high (+) and low (-), as
and Box-Behnken designs resulted in dramatic enhancement
well as two dummy variables in 20 trials (Table 2). The two
of rapamycin production and the finding of the correlation
dummy variables were used to calculate the standard error.
to several key factors, such as CaCO3 and glycerol. In
addition, this study showed an important correlation of Box-Behnken Design and Response Surface Methodology
final pH to the amount of rapamycin produced. The significant variables identified from the previous experiments
were optimized using Box-Behnken design at different levels
Materials and Methods (Table 3), while the other variables of nonsignificance were fixed
at the initial medium level. In developing the regression equation,
Microorganism and Culture Conditions the relation between the coded values and actual values are
The mutant strain of Streptomyces hygroscopicus ATCC 29253 described in Eq. (1) below: [11]
used in this study was maintained on TSB agar plates at 30°C. ( Ai – A0 )
X i = ----------------------
- (1)
Analytical chemicals were obtained from BD (San Jose, CA, USA) ∆A i

J. Microbiol. Biotechnol.
 Media Optimization of S. hygroscopicus for Rapamycin Production 1321

Table 1. High(-1) and low(+) values of the independent where Xi is the coded value of the ith variable, Ai is the actual
variables in the Plackett-Burman design. value of the ith variable, Ao is the actual value of the ith variable at
Levels of factor the center point, and ∆Ai is the step change value of the ith
Factor variable. The correlation between the response and the four variables
-1 1
were fitted to a predictive quadratic polynomial equation as
M100 (X1, g/l) 10 30 follows:
Glycerol (X2, g/l) 10 30
2
Soybean meal (X3, g/l) 1 10 Y = β0 + ∑ βi Xi + ∑ βij Xi Xj + ∑ βii Xi i = 1,2,3, ... ... k (2)

Soytone (X4, g/l) 1 10

where Y was the predicted response, β0 is the intercept term, βi is
Yeast extract (X5, g/l) 1 6.5 the linear coefficient, βii is the squared coefficient, and βij is the
(NH4)2SO4 (X6, g/l) 1 5 interaction coefficient. Xi, Xj represented the independent factors
L-Lysine (X7, g/l) 1 5 (medium component) in the form of coded values. The accuracy
and general ability of the above polynomial model could be
KH2PO4 (X8, g/l) 1 2.5
K2HPO4 (X9, g/l) 1 2.5
Table 3. Coded and real values of factors in the Box-Behnken
NaCl (X10, g/l) 1 5
design.
FeSO4·7H2O (X11, g/l) 0 0.1
Level of factor
CaCO3 (X12, g/l) 1 5 Factor
-1 0 1
MES (X13, g/l) 21 42.6 Glycerol (X2, g/l) 10 30 50
pH (X14, g/l) 5 5.5 Soytone (X4, g/l) 1 10.5 20
Dummy1 (D1, g/l) -1 1 Yeast extract (X5, g/l) 1 5.5 10
Dummy2 (D2, g/l) -1 1 CaCO3 (X12, g/l) 0 5 10

Table 2. Combinations of variables and responses in the Plackett–Burman design experiment.

Independent variables Dummy variables Rapamycin
Run
X1 X2 X3 X4 X5 X6 X7 X8 X9 X10 X11 X12 X13 X14 D1 D2 ( mg/l )
1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 29.8 ± 0.7
2 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 12.55 ± 2.15
3 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 77.35 ± 2.25
4 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 5.95 ± 0.05
5 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 41.95 ± 1.55
6 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 1 40.4 ± 18.2
7 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 -1 61.4 ± 5.5
8 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 1 30.1 ± 0.4
9 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 -1 43.7 ± 10
10 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 -1 12.15 ± 0.85
11 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 -1 43.8 ± 0.2
12 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 -1 36.95 ± 9.05
13 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 1 12.55 ± 2.85
14 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 1 42.9 ± 3.2
15 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 -1 19.45 ± 0.95
16 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 1 45.5 ± 10
17 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 1 31.3 ± 5.4
18 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 -1 30.5 ± 0.8
19 -1 1 1 -1 -1 -1 -1 1 -1 1 -1 1 1 1 1 -1 44.2 ± 12.2
20 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 -1 36.85 ± 2.95

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1322 Kim et al.

evaluated by the coefficient of determination R2 [4]. Each Optimization of Screened Medium Component Using the
experimental design was carried out in duplicate, and the mean Box-Behnken Design
values were used for further anlaysis. The factors that were The four most significant factors (glycerol, soytone,
significant at 95% confidence (p < 0.05) from the regression CaCO3, and yeast extract) among seven were selected and
analysis were considered to have greater effects on the rapamycin
examined in a Box-Behnken design in 27 runs (Table 4).
production and were further optimized by response surface
Rapamycin production was evaluated to find the combined
methodology using the Box-Behnken design.
effect of the four factors in their specific ranges. The
variables showing effect with a confidence level of 99.1%
Results
(glycerol), 100% (soytone), 99.2% (yeast extract), and 100%
(CaCO3) (Table S1) in the Plackett-Burman design were
Screening of Essential Medium Components Using the
selected, and further optimization was achived using a
Plackett–Burman Design
Box-Behnken design. A contour plot was drawn and the
Fourteen different carbon and nitrogen sources and
data revealed that CaCO3 is a critical factor to determine
inorganic salts factors were evaluated for their suitability
rapamycin productivity, whereas soytone and yeast extract
to sustain increased rapamycin production by Streptomyces
showed negative effect at certain ranges on rapamycin
hygroscopicus. The Plackett-Burman design was used for
production, and glycerol showed a positive effect (Fig. 2).
initial screening of medium components. Tables 1 and 2
show the effects of the 14 components, and Table S1 shows
Rapamycin (mg/l) =
their significant levels. Seven components, as displayed in
58.800 − 23.633 A − 16.934 B + 112.738 C − 8.534D +
Fig. 1, were found to have a significant effect on rapamycin
20.691 B2 − 18.605 D2
production (i.e., soytone, CaCO3, yeast extract, glycerol,
(A: soytone; B: CaCO3; C: glycerol; D: yeast extract) (3)
pH, FeSO4·7H2O, and soybean). The components were
screened at a confidence level of 95% on the basis of their
Rapamycin production is the predicted response and A,
effects. Soybean, FeSO4·7H2O, and the intial pH were
B, C, and D are the coded variables for soytone, CaCO3,
excluded because the effects by soybean and FeSO4·7H2O
were relatively weak and the initial pH had no significant
effects on rapamycin production. Variables showing effects
with a confidence level of 99.1% (glycerol), 100% (soytone),
99.2% (yeast extract), and 100% (CaCO3) were identified as
important factors for rapamycin production. Thereafter,
the exact optimal values for the individual factors were
determined using the Box-Behnken design.

Fig. 2. Countour plots showing the effects of independent

Fig. 1. Pareto chart of the 14-factor standard effects on variables (soytone, yeast extract, glycerol, and calcium
rapamycin production. carbonate concentration) on rapamycin production.

J. Microbiol. Biotechnol.
 Media Optimization of S. hygroscopicus for Rapamycin Production 1323

Table 4. Box-Behnken design matrix with experimental values of rapamycin production and final pH.
Run Soytone (g/l) CaCO3 (g/l) Glycerol (g/l) Yeast extract (g/l) Final pH Rapamycin (mg/l)
1 1 0 30 5.5 5.0 ± 0.0 128.4 ± 11.8
2 20 0 30 5.5 5.9 ± 0.0 60.7 ± 7.9
3 1 10 30 5.5 5.7 ± 0.1 90 ± 2.0
4 20 10 30 5.5 5.9 ± 0.0 35.7 ± 1.2
5 10.5 5 10 1 5.5 ± 0.0 33.5 ± 6.4
6 10.5 5 50 1 5.6 ± 0.1 67.5 ± 2.1
7 10.5 5 10 10 6.1 ± 0.0 17.9 ± 5.8
8 10.5 5 50 10 5.7 ± 0.0 25.2 ± 24.5
9 1 5 30 1 5.0 ± 0.1 68.9 ± 0.3
10 20 5 30 1 6.0 ± 0.1 30.8 ± 2.5
11 1 5 30 10 5.6 ± 0.1 57.8 ± 6.4
12 20 5 30 10 6.2 ± 0.1 24.0 ± 3.0
13 10.5 0 10 5.5 5.7 ± 0.0 45.2 ± 3.6
14 10.5 10 10 5.5 5.9 ± 0.2 27.5 ± 0.3
15 10.5 0 50 5.5 5.4 ± 0.0 101 ± 2.1
16 10.5 10 50 5.5 6.1 ± 0.1 52.5 ± 2.6
17 1 5 10 5.5 6.0 ± 0.1 75.2 ± 0.9
18 20 5 10 5.5 6.6 ± 0.0 12.5 ± 0.2
19 1 5 50 5.5 5.4 ± 0.1 66.8 ± 24.8
20 20 5 50 5.5 6.1 ± 0.1 39.7 ± 4.1
21 10.5 0 30 1 5.4 ± 0.1 116.1 ± 4.9
22 10.5 10 30 1 5.7 ± 0.1 66.0 ± 4.4
23 10.5 0 30 10 6.2 ± 0.0 89.6 ± 1.0
24 10.5 10 30 10 5.9 ± 0.1 66.0 ± 0.9
25 10.5 5 30 5.5 5.5 ± 0.0 73.2 ± 0.9
26 10.5 5 30 5.5 5.5 ± 0.0 71.5 ± 1.3
27 10.5 5 30 5.5 5.6 ± 0.1 68.0 ± 1.4

glycerol, and yeast extract respectively (Eq. (3)). Soytone,

CaCO3, and yeast extract had negative effects in the
rapamycin production media, whereas glycerol showed
positive effects on rapamycin productivity. The expected
maximum rapamycin production was 130.4 mg/l using
media containing glycerol 36.2 g/l, soytone 1 g/l, yeast
extract 1 g/l, and no CaCO3.

Effect of pH on Rapamycin Production

The effect of the initial pH value on rapamycin
production was examined in the Plackett-Burman design.
The effects from initial pH were smaller than the other
significant factors, and no significant effects were observed
when the initial pH was between 6 and 8 in the flask trials
[24]. Thus, we excluded the initial pH factor from the effect Fig. 3. Correlation of final pH and rapamycin production on
factors. However, the final pH was observed for correlation data used for the Plackett-Burman design.

October 2014 ⎪ Vol. 24 ⎪ No. 10

1324 Kim et al.

production reached 220.7 ± 5.7 mg/l in the optimized media

from S. hygroscopicus mutant, whereas 151.9 ± 22.6 mg/l
rapamycin production was recorded in the unoptimized
media. For the wild strain, 76.2 ± 4.7 and 37.5 ± 2.8 mg/l
rapamycin production were observed in optimized and
unoptimized media, respectively (Fig. 5). We observed an
increase in the production of rapamycin by 5.88-fold
compared with-the wild type S. hygroscopicus.

Discussion

Rapamycin is a potent immunosuppressive secondary

metabolite that has been increasingly of interest for clinical
treatments. However, the low titers of rapamycin limited
Fig. 4. Time course data of pH during rapamycin production its availability for expanded industrial use [19]. The
in optimized and unoptimized media for wild-type and present report is an attempt to formulate a glycerol-based
mutant-type Streptomyces hygroscopicus. medium for rapamycin production with S. hygroscopicus. A
systematic study of rapamycin improvement through
between the pH and production of rapamycin (Fig. 3). We medium optimization, and the effects of various medium
found rapamycin production increased linearly with the components at different concentrations were investigated
decrease in pH. For this reason, final pH was statistically a using a Plackett-Burman experiment. From the 14 factors,
significant factor; namely, a decrease in pH levels leads to soytone, glycerol, CaCO3, and yeast extract were selected
an increase in rapamycin production (Fig. 4). for their effect on rapamycin production in a Box-Behnken
design for further optimization. We found that CaCO3,
pH = 0.0154 (rapamycin) + 5.963 (4) soytone, and yeast extract showed negative effects on
rapamycin production, whereas the glycerol showed a
rapamycin = 64.94 pH – 387.21 (4.8 < pH < 6.3) (5) positive effect. CaCO3 modulated the dissociated-
undissociated equilibrium and prevented a reduction in
CaCO3 is a determining factor for the change of pH, and pH during the fermentation [15]. Omission of CaCO3 and a
so the decrease of rapamycin production with an increase lower amount of nitrogen resulted in the medium having a
in pH indicated that CaCO3 is a critical and negative factor lower final pH [13]. The glycerol showed positive effect,
for rapamycin production. The maximum rapamycin and importantly the final pH also showed correlation with
rapamycin production. Glycerol is potentially also an
economical feedstock for fermentation, and it can be
consumed more quickly after adding ammonium sulfate in
the rapamycin biosynthesis phase, which will lead to an
increase of the pool of rapamycin biosynthesis. This unique
characteristic of glycerol offers a tremendous opportunity
for its biological conversion to valuable products at higher
yield. The total amount of rapamycin produced was
correlated to final pH and it was found that the acidic pH
might have triggered initiation of the stationary or
secondary metabolite production phase [7]. Although the
exact mechanism was not revealed, different metabolites
related to the TCA cycle, fatty acids metabolism, and fatty
acid degradation seemed to trigger the drop in pH,
resulting in pH shock and contributing to activation of the
Fig. 5. Comparison of rapamycin production by Streptomyces TCA cycle and production of secondary metabolites [7, 23].
hygroscopicus in optimized and unoptimized media. Finally, it is expected that an acidic pH strongly promoted

J. Microbiol. Biotechnol.
 Media Optimization of S. hygroscopicus for Rapamycin Production 1325

production of rapamycin, as the pH can affect the 1995. Glycerol effect on spiramycin production and valine
production of rapamycin complex by changing the electron catabolism in Streptomyces ambofaciens. Curr. Microbiol. 31:
interaction of the rings. 304-311.
In conclusion, this research is a systematic optimization 11. Ma FX, Kim JH, Kim SB, Seo YG, Chang YK, Hong SK, Kim
CJ. 2008. Medium optimization for enhanced production of
for rapamcyin production by a S. hygroscopicus mutant. By
rifamycin B by Amycolatopsis mediterranei S699: combining a
applying statistical design, we have desgined a medium for
full factorial design and a statistical approach. Process
rapamycin production, resulting in an increase of rapamycin
Biochem. 43: 954-960.
production by nearly 40% compared with an unoptimized 12. Osman MI. 1997. Exploring a mixture of distributions using
production medium. Minitab. Comput. Biol. Med. 27: 223-232.
13. Papagianni M, Wayman F, Mattey M. 2005. Fate and role of
Acknowledgments ammonium ions during fermentation of citric acid by
Aspergillus niger. Appl. Environ. Microbiol. 71: 7178-7186.
This work was supported by the faculty research fund of 14. Qian W, Yu C, Qin H, Liu X, Zhang A, Johansen IE, Wang D.
2007. Molecular and functional analysis of phosphomannomutase
Konkuk University in 2012.
(PMM) from higher plants and genetic evidence for the
involvement of PMM in ascorbic acid biosynthesis in
References Arabidopsis and Nicotiana benthamiana. Plant J. 49: 399-413.
15. Raganati F, Olivieri G, Procentese A, Russo ME, Salatino P,
1. Chen X, Wei P, Fan L, Yang D, Zhu X, Shen W, et al. 2009. Marzocchella A. 2013. Butanol production by bioconversion
Generation of high-yield rapamycin-producing strains through of cheese whey in a continuous packed bed reactor.
protoplasts-related techniques. Appl. Microbiol. Biotechnol. 83: Bioresour. Technol. 138: 259-265.
507-512. 16. Ritacco FV, Graziani EI, Summers MY, Zabriskie TM, Yu K,
2. Da Silva GP, Mack M, Contiero J. 2009. Glycerol: a promising Bernan VS, et al. 2005. Production of novel rapamycin
and abundant carbon source for industrial microbiology. analogs by precursor-directed biosynthesis. Appl. Environ.
Biotechnol. Adv. 27: 30-39. Microbiol. 71: 1971-1976.
3. Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, 17. Sayyad SA, Panda BP, Javed S, Ali M. 2007. Optimization of
Flurkey K, et al. 2009. Rapamycin fed late in life extends nutrient parameters for lovastatin production by Monascus
lifespan in genetically heterogeneous mice. Nature 460: 392- purpureus MTCC 369 under submerged fermentation using
395. response surface methodology. Appl. Microbiol. Biotechnol.
4. Jeon JM, Rajesh T, Song E, Lee HW, Yang YH. 2013. Media 73: 1054-1058.
optimization of Corynebacterium glutamicum for succinate 18. Sehgal SN, Baker H, Vezina C. 1975. Rapamycin (AY-
production under oxygen-deprived condition. J. Microbiol. 22,989), a new antifungal antibiotic. II. Fermentation, isolation
Biotechnol. 23: 211-217. and characterization. J. Antibiot. (Tokyo) 28: 727-732.
5. Kahan BD, Camardo JS. 2001. Rapamycin: clinical results 19. Singh N, Rai V. 2012. Improved antimicrobial compound
and future opportunities. Transplantation 72: 1181-1193. production by a new isolate Streptomyces hygroscopicus
6. Khaw LE, Bohm GA, Metcalfe S, Staunton J, Leadlay PF. MTCC 4003 using Plackett-Burman design and response
1998. Mutational biosynthesis of novel rapamycins by a surface methodology. Bioinformation 8: 1021-1025.
strain of Streptomyces hygroscopicus NRRL 5491 disrupted in 20. Wang L. 2001. UV mutagenesis in Escherichia coli K-12: cell
rapL, encoding a putative lysine cyclodeaminase. J. Bacteriol. survival and mutation frequency of the chromosomal genes
180: 809-814. lacZ, rpoB, ompF, and ampA. J. Exp. Microbiol. Immunol. 45:
7. Kim YJ, Song JY, Moon MH, Smith CP, Hong SK, Chang 192-197.
YK. 2007. pH shock induces overexpression of regulatory 21. West NP, Jungnitz H, Fitter JT, McArthur JD, Guzman CA,
and biosynthetic genes for actinorhodin production in Walker MJ. 2000. Role of phosphoglucomutase of Bordetella
Streptomyces coelicolor A3(2). Appl. Microbiol. Biotechnol. 76: bronchiseptica in lipopolysaccharide biosynthesis and virulence.
1119-1130. Infect. Immun. 68: 4673-4680.
8. Kojima I, Cheng YR, Mohan V, Demain AL. 1995. Carbon 22. Xu ZN, Shen WH, Chen XY, Lin JP, Cen PL. 2005. A high-
source nutrition of rapamycin biosynthesis in Streptomyces throughput method for screening of rapamycin-producing
hygroscopicus. J. Ind. Microbiol. 14: 436-439. strains of Streptomyces hygroscopicus by cultivation in 96-well
9. Lee MS, Kojima I, Demain AL. 1997. Effect of nitrogen microtiter plates. Biotechnol. Lett. 27: 1135-1140.
source on biosynthesis of rapamycin by Streptomyces 23. Yang YH, Song E, Lee BR, Kim EJ, Park SH, Kim YG, et al.
hygroscopicus. J. Ind. Microbiol. Biotechnol. 19: 83-86. 2010. Rapid functional screening of Streptomyces coelicolor
10. Lounes A, Lebrihi A, Benslimane C, Lefebvre G, Germain P. regulators by use of a pH indicator and application to the

October 2014 ⎪ Vol. 24 ⎪ No. 10

1326 Kim et al.

MarR-like regulator AbsC. Appl. Environ. Microbiol. 76: 3645- metabolic pathway-based mutagenesis and further titer
3656. improvement with fed-batch bioprocess optimization. Biotechnol.
24. Yen HW, Li FT, Wong CL, Chang JS. 2013. The pH effects Bioeng. 107: 506-515.
on the distribution of 1,3-propanediol and 2,3-butanediol 26. Zou X, Li WJ, Zeng W, Hang HF, Chu J, Zhuang YP, Zhang
produced simultaneously by using an isolated indigenous SL. 2012. Biochemical parameters of Saccharopolyspora erythraea
Klebsiella sp. Ana-WS5. Biopross Biosyst. Eng. 37: 425-431. during feeding ammonium sulphate in erythromycin
25. Zhu X, Zhang W, Chen X, Wu H, Duan Y, Xu Z. 2010. biosynthesis phase. Prikl. Biokhim. Mikrobiol. 49: 190-196.
Generation of high rapamycin-producing strain via rational

J. Microbiol. Biotechnol.