ORIGINAL RESEARCH PAPER
Characterization of a flavin-containing monooxygenasefrom Corynebacterium glutamicum and its applicationto production of indigo and indirubin
Sisi Patricia Lolita Ameria • Hye Sook Jung •
Hee Sook Kim • Sang Soo Han • Hak Sung Kim •
Jin Ho Lee
Received: 8 January 2015 / Accepted: 23 March 2015 / Published online: 8 April 2015
� Springer Science+Business Media Dordrecht 2015
Abstract
Objective To examine the role of a gene encoding
flavin-containing monooxygenase (cFMO) from
Corynebacterium glutamicum ATCC13032 when
cloned and expressed in Escherichia coli for the
production of indigo pigments.
Results The blue pigments produced by recombinant
E. coli were identified as indigo and indirubin. The
cFMO was purified as a fused form with maltose-
binding protein (MBP). The enzyme was optimal at
25 �C and pH 8. From absorption spectrum analysis,
the cFMO was classified as a flavoprotein. FMO
activity was strongly inhibited by 1 mM Cu2? and
recovered by adding 1–10 mM EDTA. The enzyme
catalyzed the oxidation of TMA, thiourea, and
cysteamine, but not glutathione or cysteine. MBP-
cFMO had an indole oxygenase activity through
oxygenation of indole to indoxyl. The recombinant
E. coli produced 685 mg indigo l-1 and 103 mg
indirubin l-1 from 2.5 g L-tryptophan l-1.
Conclusion The results suggest the cFMO can be
used for the microbial production of both indigo and
indirubin.
Keywords Corynebacterium glutamicum � Flavin-containing monooxygenase � Indigo � Indirubin �Maltose-binding protein � Monooxygenase
Introduction
Indigo is a blue dye extracted from plants and is
primarily used for the production of denim cloth for
blue jeans (Ensley et al. 1983). This dye is presently
produced by chemical synthesis. Indirubin, a 3,20-bisindole isomer of indigo, is the active constituent of
a traditional Chinese medicine, Danggui Longhui
Wan, which is a mixture of herbal plants, and is
currently used for treatment of chronic myelocytic
leukemia (Eisenbrand et al. 2004). Despite the poten-
tial of indirubin as a therapeutic agent, the develop-
ment of indirubin-based therapy has been impeded by
its low availability (Moon et al. 2006).
Electronic supplementary material The online version ofthis article (doi:10.1007/s10529-015-1824-2) contains supple-mentary material, which is available to authorized users.
S. P. L. Ameria
Food Technology Department, Faculty of Food Science &
Technology, Universitas Pelita Harapan, 1100, Jl.M.H.
Thamrin Boulevard Raya, Tangerang 15811, Indonesia
H. S. Jung � H. S. Kim � J. H. Lee (&)
Department of Food Science & Biotechnology,
Kyungsung University, 309, Suyeong-ro, Nam-gu,
Busan 608-736, Republic of Korea
e-mail: [email protected]
S. S. Han � H. S. KimDepartment of Biological Sciences, Korea Advanced
Institute of Science and Technology, 373-1, Gusung-dong,
Yusung-gu, Daejon 305-701, Republic of Korea
123
Biotechnol Lett (2015) 37:1637–1644
DOI 10.1007/s10529-015-1824-2
Production of indigoid compounds, indigo and
indirubin, was attempted by several approaches such
as chemical synthesis, extraction from plants, such as
Indigofera and Polygonum tinctorium, bioconversion
of indican into indirubin by non-recombinant Escher-
ichia coli (Lee et al. 2011), and biological synthesis
from L-tryptophan by various oxygenases (Ensley
et al. 1983; Lu andMei 2007). Of these,much attention
has been paid to microbial production of indigo and
indirubin based on multi- and single-component
oxygenases including naphthalene dioxygenase, phe-
nol hydroxylase, flavin-containing monooxygenases
(FMO), and indole oxygenases (Choi et al. 2003; Kang
andLee 2009; Lim et al. 2005; Singh et al. 2010).Most
bacteria expressing these oxygenases were shown to
produce indigo as a major product, whereas accumu-
lated a small amount of indirubin (Han et al. 2012; Lim
et al. 2005; Singh et al. 2010).
FMOs (EC 1.14.13.8) catalyze the conversion of
nucleophilic hetero-atom compounds, such as nitro-
gen, sulfur or phosphorus, into N-oxides, S-oxides or
P-oxides, respectively (Cashman 2005; van Berkel
et al. 2006). FMOs have been found and characterized
in mammals, plants, invertebrates, and yeast (Cash-
man 1995; Lomri et al. 1992; Suh et al. 1996; van
Berkel et al. 2006). An FMO from Methylophaga
aminisulfidivorans MPT (mFMO) was isolated and
characterized its biochemical properties (Choi et al.
2003). The mFMO catalyzed the hydroxylation of
indole as well as N-containing amines, and E. coli
containing mFMO produced 920 mg indigo l-1 and
5 mg indirubin -1 in a 5 l fermenter from 2 g L-
tryptophan l-1 (Han et al. 2008). Although some
bacterial FMOs from Mesorhizobium loti and Sphin-
gomonas wittichii have been identified, little informa-
tion is known about the biochemical properties and
biocatalytic potential of bacterial FMOs in general
(Choi et al. 2003; Singh et al. 2010).
Corynebacterium glutamicum is widely used in the
industrial production of many amino acids as host cells
and has been engineered to produce several valuable
bio-based chemicals and proteins (Lee 2014). Since a
putative FMO (NCgl1096, cFMO) showing a high
sequence homology to mFMO was known in
C. glutamicum, we chose this cFMO in this study.
Thus, the gene encoding aCorynebacterium FMOwas
cloned in E. coli and the purified cFMO has been
characterized. The indigoid-forming ability of recom-
binant E. coli cells was then evaluated.
Materials and methods
Bacterial strains, plasmids, and medium
Bacterial strains and plasmids used in this study are
listed in Supplementary Table 1. Wild-type C. glu-
tamicum ATCC13032 was used to provide the DNA
template for cloning of fmo gene. Escherichia coli
Top10 and W3110 were employed to host strains for
general DNA manipulation and indigoid production,
respectively. Plasmids pKK223-3 and pMAL-c2x
were used for the expression and purification of
cFMO, respectively. Escherichia coli was grown in
lysogeny broth (LB) medium (10 g tryptone l-1, 5 g
yeast extract l-1, and 10 g NaCl l-1) with 100 lgampicillin ml-1 when necessary. Expression of cFMO
was induced by adding 0.1 mM IPTG into culture
broth when the OD600 was 0.5–0.6.
Construction of expression vectors
A putative flavin-containing monooxygenase 3
(NCgl1096) from C. glutamicum was cloned and
expressed in pKK223-3. For amplification of fmo,
primers 1 (50-CCCGGAATTCATGGAGATGGTTATGAAGAA-30) and 2 (50-CCCCAAGCTTTTAGGCTTTATCGCGGACTT-30) were used. About 1.4 kb of
the amplified fragment was double-digested with
EcoRI and HindIII, ligated with EcoRI/HindIII-
cleaved pKK223-3, and transformed into E. coli
Top10. The resulting plasmid was designated pPIO1.
To purify and characterize cFMO fused to maltose-
binding protein (MBP), the 1.4 kb fmo fragment of
pPIO1 was cut with EcoRI and HindIII and subcloned
into the same restriction sites of pMAL-c2x to yield
plasmid pMCF14.
Identification and analysis of indigoid compounds
Escherichia coli W3110 harboring pPIO1 was culti-
vated in 20 ml LB medium with 5 g L-tryptophan l-1
at 32 �C with IPTG induction, and culture broth was
taken for analysis by TLC. Briefly, 1 ml culture broth
was centrifuged at 10,0009g for 1 min, the cells were
washed three times with distilled water, suspended in
200 ll dimethylsulfoxide, and the extract applied to a
silica gel plate (Merck; Kang and Lee 2009). HPLC
was used to identify and quantify indigo and indirubin,
which were detected at 600 and 540 nm, respectively
1638 Biotechnol Lett (2015) 37:1637–1644
123
(Kang and Lee 2009). To confirm the identity of the
two constituents, the indigoid mixture was purified by
silica gel chromatography according to Lim et al.
(2005) and then analyzed by GC–MS. The purified
compound was diluted in chloroform and injected to
Agilent 5973 N GC–MS (USA), equipped with a
quadrupole mass filter and VF-1 ms capillary column
(60 m 9 0.32 mm) with a film thickness of 0.25 lm(Varian, USA).
Purification of the fusion protein, maltose-binding
protein (MBP)-cFMO
To express and purify MBP-cFMO, the recombinant
E. coliwith pMCF14 was induced with IPTG and then
taken from cultures after incubation for 16 h at 28 �C.Cells were harvested by centrifugation at 50009g for
10 min, washed with column buffer (20 mM Tris/
HCl, 200 mM NaCl, pH 7.4), resuspended with lysis
buffer (20 mM Tris/HCl, 0.2 M NaCl, 1 mM EDTA,
0.5 % Triton X-100, 1 mM PMSF, 10 lM FAD, pH
7.4), and lysed by sonication. After centrifugation at
13,0009g for 30 min, the supernatant was filtered
using a 0.2 lm syringe filter and loaded onto affinity
column containing amylose gel equilibrated with
column buffer. MBP-cFMO was eluted from the
column with elution buffer (10 mMmaltose in column
buffer). The purity of MBP-cFMO was detected by
SDS-PAGE analysis. Protein concentration was de-
termined by Bradford method (Bio-Rad) with bovine
serum albumin as the standard.
Enzyme assay, absorption spectrum analysis,
and measurement of FAD concentration
NADPH oxidase activity was measured spectropho-
tometrically using N- and S-containing substrates at
room temperature and calculated by subtracting the
futile activity of NADPH oxidase which was deter-
mined in the absence of substrate at the same assay
condition. The reaction mixture, 1 ml, consisted of
0.1 M potassium phosphate (pH 8.0), 200 lMNADPH, 2 lM FAD, and 40 lg MBP-cFMO. One
unit of NADPH oxidase activity was defined as the
amount of enzyme required to oxidation of 1 nmol
NADPH per min per mg protein at 340 nm in the
presence of substrate. The Km and kcat values toward
several substrates were calculated based on the
Lineweaver–Burk plots. Indole oxygenase activity
was measured by the spectrofluorescence of indoxyl
with excitation at 365 nm and emission at 470 nm
(Cho et al. 2011). All enzyme assays were performed
in triplicate experiments. The reduced form of purified
MBP-cFMO was prepared by addition of 0.5 mM
sodium hydrosulfite, and then absorption spectra of
MBP-cFMO in the oxidized and reduced states were
scanned from 600 to 300 nm. The bound FAD
concentration in MBP-FMO was measured according
to the previously described method (Choi et al. 2003).
Production and analysis of indigoids
Indigoid production was performed in LB medium
supplemented with 2.5 g L-tryptophan l-1 in a shaking
incubator at 32 �C for 48 h using recombinant E. coli
cells. Flask fermentation was performed in triplicate.
The amounts of indigo, indirubin, and L-tryptophan
were measured by HPLC (Kang and Lee 2009).
Results and discussion
Cloning and expression of a gene encoding FMO
from C. glutamicum
To construct recombinant E. coli expressing a FMO
that catalyzes oxygenation of indole, the gene encod-
ing the putative FMO of C. glutamicum was cloned
into an expression vector of E. coli, and the resulting
plasmid was designated pPIO1. The tentative FMO is
composed of 470 amino acids, with a calculated
molecular mass of 54 kDa. Expression of the FMO in
E. coli was tested, and a band appeared at the
molecular mass of about 55 kDa from the crude
extracts of corresponding host cells (Fig. 1a). The
recombinant cells grown in a LB plate with indole or
L-tryptophan were deep blue, which implies the
expressed putative cFMO in E. coli mediates the
production of indigoid compounds from indole.
Analysis and identification of indigoid compounds
The deep blue products extracted from E. coliWCO21
were analyzed by TLC. Two spots, one blue and one
red, were appeared and migration profiles of them
were the same as those of synthetic indigo and
indirubin, respectively (Fig. 2). Analyses by spec-
trophotometry and HPLC revealed that the purified
Biotechnol Lett (2015) 37:1637–1644 1639
123
blue and red compounds display maximum ab-
sorbance peaks at 610 and 540 nm, respectively, and
have the same retention time as the standard indigo
and indirubin, respectively (Choi et al. 2003; Kang and
Lee 2009). Molecular weights of the purified com-
pounds were determined to be 262 Da by GC–MS,
which correspond to the exact molecular weights of
standard indigo and indirubin, respectively. These
results demonstrate that the deep blue pigments
produced by E. coli expressing cFMO are indigo and
indirubin.
Sequence analysis of cFMO
To find homologous proteins, a BLAST search was
performed using the cFMO amino acid sequence as a
query which was compared with entries in the NCBI
database (non-redundant protein sequences). The
cFMO showed high sequence homologies to the
sequences of experimentally identified FMO ofMethy-
lophaga aminisulfidivorans MPT (JC7986, 72 %) and
uncharacterized putative flavin-containing monooxy-
genases or oxidoreductases from Corynebacterium
efficiens YS-314 (EEW50141, 90 %), Arthrobacter
arilaitensis Re117 (CBT75263, 81 %), and Dietzia
cinnamea P4 (EFV92056, 78 %). However, relatively
low homologies (45–51 %) were found for well-
known FMOs from human, mouse, and yeast (Lomri
et al. 1992; Suh et al. 1996). Multiple sequence
alignments among these proteins indicate that two
commonmotif sequences including a FMO-identifying
sequence motif (FxGxxxHxxx(Y/F)) and a Rossmann
fold for FAD (GxGxxG) were well conserved in
Corynebacterium FMO (Fig. 3; Choi et al. 2003;
Fraaije et al. 2002; Kleiger and Eisenberg 2002).
However, a Rossmann fold for NADPH showed some
variation depending on FMO homologs. It was
GxGxxG in FMOs from human, porcine, and yeast,
whereas it was GxSxxA in FMOs from Corynebac-
terium and most other bacteria. Also, two pivotal
residues, Tyr-212 and Arg-234, regarding the indole
oxygenation and the preferential binding to NADPH
rather than NADH, respectively, were strictly con-
served (Cho et al. 2011). Thus, it is inferred that the
225
150
100
75
50
35
25
15
(a) (b)
Fig. 1 Expression (a) and purification (b) of flavin-containingmonooxygenase from C. glutamicum ATCC13032. a cFMO
was expressed in E. coli Top10 and analyzed by SDS-PAGE
using crude extract. Lanes 1, E. coli Top10/pKK223-3; 2, E. coli
Top10/pPIO1. b MBP-cFMO was purified by amylose resin
column and analyzed by SDS-PAGE. Lane 3 shows E. coli
Top10/pMCF14
Fig. 2 Analysis of indigoid pigments by thin-layer chromatog-
raphy (TLC). Lanes 1, 2, and 3 indicated pigments extracted
from E. coliWCO21, synthetic (standard) indigo, and synthetic
indirubin
1640 Biotechnol Lett (2015) 37:1637–1644
123
cFMO belongs to NADPH-dependent flavin-contain-
ing monooxygenase with indole oxygenation activity
based on sequence homology analysis.
Characterization of cFMO
Since the cFMO domain of purified MBP-cFMO
(Fig. 1b) was degraded during treatment of Factor Xa,
we determined biochemical properties of cFMO fused
to MBP. The cFMO showed a pH optimum at 8.0 in
0.1 M potassium phosphate buffer; however, the
activity was decreased to about 2.5–7 % at pH 8.0 in
Tris/HCl or Tricine buffer (Supplementary Fig. 1a).
The enzyme activity toward TMA was highest at
25 �C and was reduced significantly with increasing
temperature (Supplementary Fig. 1b). The absorption
spectra of MBP-cFMO in the oxidized (two peaks at
370 and 455 nm) and reduced forms displayed typical
pattern of flavoproteins (Supplementary Fig. 2; Choi
et al. 2003; Suh et al. 1996). The amount of bound
Fig. 3 Multiple sequence alignment of flavin-containing
monooxygenase from C. glutamicum ATCC13032 with FMOs
from different sources. The FMO sources are as follows: 1, C.
glutamicumATCC13032; 2,C. efficiensYS314; 3,Arthrobacter
arilaitensis Re117; 4, Dietzia cinnamea P4; 5, Methylophaga
aminisulfidivoransMPT. The identical and similar residues in all
of the proteins are shown as a box and a gray background,
respectively. The symbols filled circle, asterisk, and open
diamond indicate the conserved residues of Rossmann fold for
FAD, FAD-identifying motif, and Rossmann fold for NADPH.
The symbols cross in a circle and inverted triangle display
tyrosine-212 and arginine-234 residues in C. glutamicum,
respectively
Biotechnol Lett (2015) 37:1637–1644 1641
123
FAD on 1 mol MBP-cFMO was estimated to be about
0.475 mol. These results demonstrate cFMO is a
member of the FAD-containing flavoprotein family.
When the effect of several metal ions was examined.
Using TMA as a substrate, addition of 1 mM Na?,
K?, Ca2?, Co2?, Fe2?, Mn2?, Mg2?, and Zn2?
showed about 60–70 % of initial activity, whereas
FMO activity was strongly inhibited by Cu2? which
was recovered by addition of 1–10 mM EDTA. The
FMO family is involved in the oxidation of heteroa-
toms, such as N, S, Por Se, in a range of structurally
diverse compounds (van Berkel et al. 2006). The
cFMO exhibited a distinct activity toward N-contain-
ing TMA and S-containing thiourea or cysteamine,
whereas no activity was observed for S-containing
glutathione or cysteine (Table 1). The Km for TMA
and cysteamine were about 30–33-fold higher than
those of the enzyme fromM. aminisulfidivorans, while
similar levels of Km for thiourea were observed in
FMOs from both bacterial strains.
To check if the cFMO was directly associated with
the production of indigoid compounds, we determined
the indole oxygenase activity of MBP-FMO. The
cFMO exhibited the indoxyl production activity, and
its activity was enhanced by the increase of indole
concentration (Fig. 4). Collectively, based on the
above results, cFMO is able to oxidize not only
N-atom in TMA and S-atom in thiourea or cysteamine
but also C-atom in indole.
Production of indigo and indirubin by recombinant
E. coli
We conducted indigoid production from L-tryptophan
using the recombinant E. coliWCO21 which is able to
convert L-tryptophan to indole and pyruvate by an
endogeneous tryptophanase. Indigo and indirubin
were simultaneously produced after 12 h cultivation,
and gradually increased with cultivation time (Fig. 5).
Table 1 Kinetic parameters for MBP-FMO from C. glutamicum using various substrates
Substrate Km (mM) Vmax (nmol/min/mg protein) Kcat (min-1) Kcat/Km (min-1 mM-1)
Trimethylamine 0.58 1610 156 270
Thiourea 0.38 415 40 105
Cysteamine 6 1140 110 18
Activities with cysteine and glutathione were not detectable
0
5
10
15
20
25
30
0 100 200 300 400 500
Fluo
resc
ence
(Ex,
365
nm
; Em
, 470
nm
)
Time (s)
indole 5 mM
indole 1 mM
without indole
Fig. 4 Indole oxygenase activity of MBP-cFMO. Indole
oxygenase activity was measured by the fluorescence of
produced indoxyl in the presence of 1 and 5 mM indole,
respectively
0
200
400
600
800
1000
0
500
1000
1500
2000
2500
3000
0 10 20 30 40 50
Try
ptop
han
(mg
Indi
go, i
ndir
ubin
(m
g
Culture time (h)
residual tryptophan
indigo
indirubin
l-1) l-1
)Fig. 5 Production of indigo and indirubin in recombinant
E. coliWCO21. Recombinant strain was cultured in LBmedium
with 2.5 g tryptophan l-1 for 48 h at 32 �C. Indigo, indirubin,and tryptophan concentrations were determined by HPLC
analyses
1642 Biotechnol Lett (2015) 37:1637–1644
123
E. coli cells expressing cFMO in LB medium with
2.5 g L-tryptophan l-1 produced 685 mg indigo l-1
and 103 mg indirubin l-1 while consuming 1.84 g
L-tryptophan l-1 after 48 h culture in a shake-flask
fermentation. The total molar conversion yield for
indigoid from L-tryptophan was about 67 %. Many
recombinant cells can produce about 25–920 mg
indigo l-1 as a major product with indirubin as a
minor component by expressing several types of
oxygenases (Ensley et al. 1983; Han et al. 2011, 2008;
Lu and Mei 2007). Our result suggests the cFMO
would be applicable for the microbial production of
indigo following process optimization in a large-scale
fermentor. Moreover, the production of 103 mg
indirubin l-1 in shake-flasks is the second highest
level when compared with a previous report in which
an E. coli expressing mFMO produced 224 mg
indirubin l-1 by adding 0.36 g L-cysteine l-1 in a
10 l fermentor (Han et al. 2012). We anticipate that it
will be possible to produce higher amounts of
indirubin through a high-cell density culture in a 5 l
fermentor, which will be meaningful to solve the
problem of low availability in developing indirubin-
based therapeutic agents.
Conclusion
The Corynebacterium FMO was purified as a MBP-
fused form and its biochemical properties were
characterized. The enzyme was classified as a typical
flavoprotein and oxidized not only the N in TMA and
the S in thiourea or cysteamine but also a C in indole.
Application of an E. coli whole-cell biocatalyst
expressing cFMO in LB medium with 2.5 g
L-tryptophan l-1 led to the production of 685 mg
indigo l-1 and 103 mg indirubin l-1. This result
provides a possible way to improve production of
indigo and indirubin by expressing C. glutamicum
FMO and media optimization, and we are expected to
expand for mass production of these chemicals in the
future.
Acknowledgments This research was supported by Basic
Science Research Program through the National Research
Foundation of Korea (NRF) funded by the Ministry of
Education, Science andTechnology (NRF-2012R1A1A2007229).
Supporting information Supplementary Table 1—The bac-
terial strains and plasmids used in this study.
Supplementary Figure 1—Effect of pH (a) and temperature
(b) on the NADPH oxidase activity of purified MBO-Cfmo.
Supplementary Figure 2—Absorption spectra of purified
MBP-cFMO.
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