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: jhlee83@ks.ac.kr

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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