Biosynthesis of indigo in Escherichia coli expressing self-sufficient CYP102A from Streptomyces cattleya

Article abstract of DOI:10.1016/j.dyepig.2017.01.029

Cytochrome P450 monooxygenases (CYP) are a superfamily of heme-thiolate proteins which catalyze the incorporation of oxygen atoms into substrates. Here, a self-sufficient CYP102A from Streptomyces cattleya (CYP102A_scat) was cloned, produced recombinantly in Escherichia coli strain BL21 (DE3), and the characteristic features were investigated. However, unlike other self-sufficient CYP102A enzymes that have been reported, CYP102A_scat was found to be able to catalyze intracellular hydroxylation of indole molecules with 3-C specific regioselectivity. Consequently, E.?coli strains producing CYP102A_scat could synthesize approximately 1.0?g/L of indigo in LB media. Optimization of indigo synthesis was investigated through additional feeding of indole precursors such as glucose, L-tryptophan, and indole. Indigo production reached up to 3.8?±?0.1?g/L by adding 20?μM of extracellular indole and 0.2?mM of L-tryptophan to the LB media. To our knowledge, this is a record and the highest yield achieved so far.

Full text of DOI:10.1016/j.dyepig.2017.01.029

Dyes and Pigments 140 (2017) 29e35
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Biosynthesis of indigo in Escherichia coli expressing self-sufficient
CYP102A from Streptomyces cattleya
Hee-Jung Kim a, Seyoung Jang a, Joonwon Kim b, Yung-Hun Yang c, Yun-Gon Kim d,
,
*
Byung-Gee Kim b, Kwon-Young Choi a
a Department of Environmental Engineering, College of Engineering, Ajou University, Suwon, Gyeonggi-do, South Korea
b Department of Chemical and Biological Engineering, Seoul National University, Seoul, South Korea
c Department of Microbial Engineering, College of Engineering, Konkuk University, Seoul, South Korea
d Department of Chemical Engineering, College of Engineering, Soongsil University, Seoul, South Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Cytochrome P450 monooxygenases (CYP) are a superfamily of heme-thiolate proteins which catalyze the
incorporation of oxygen atoms into substrates. Here, a self-sufficient CYP102A from Streptomyces cattleya
(CYP102A_scat) was cloned, produced recombinantly in Escherichia coli strain BL21 (DE3), and the
characteristic features were investigated. However, unlike other self-sufficient CYP102A enzymes that
have been reported, CYP102A_scat was found to be able to catalyze intracellular hydroxylation of indole
molecules with 3-C specific regioselectivity. Consequently, E. coli strains producing CYP102A_scat could
synthesize approximately 1.0 g/L of indigo in LB media. Optimization of indigo synthesis was investigated
Received 5 May 2016
Received in revised form
10 December 2016
Accepted 12 January 2017
Available online 16 January 2017
Keywords:
through additional feeding of indole precursors such as glucose,
duction reached up to 3.8 0.1 g/L by adding 20 M of extracellular indole and 0.2 mM of
the LB media. To our knowledge, this is a record and the highest yield achieved so far.
L-tryptophan, and indole. Indigo pro-
Cytochrome P450 monooxygenases
Self-sufficient CYP102A
Indigo
m
L-tryptophan to
© 2017 Elsevier Ltd. All rights reserved.
Eco-friendly dye
Indole
L-tryptophan
1. Introduction
glycine provided
a
new and economically attractive route.
Approximately 20 million kilograms of indigo are produced annu-
ally via this synthetic route, and that indigo is used mainly for
dyeing blue jeans, and as a colorant in the food industry. Although
indigo has a low oral toxicity, with an LD50 of 5.0 g/kg in mammals,
large spills of blue cloth dyes, which include indigo, are nonetheless
hazardous because they contain harmful chemical byproducts [5,6].
With the increasing need to develop eco-friendly processes and
the growing interest in natural pigments, various attempts have
been made to synthesize natural dyes and pigments biologically.
One of the most promising of such processes is the use of microbial
enzymes within engineered metabolic pathways for the synthesis
of indigo, using any of several renewable biomass sources as a
feedstock [7e10]. Compared to production routes based on syn-
thetic chemistry, this biological route is relatively simple and fast.
Given appropriate biologically-sourced starting-chemicals, biolog-
ical conversions such as biotransformation and enzymatic modifi-
cations of structure can result in the effective synthesis of indigo
dye with excellent production yields [11,12]. For example, several
attempts for indigo production using flavin-containing mono-
oxygenases (FMO) have been reported [13,14]. Recent research on
Indigo-blue, which has a deep dark-blue color, is one of the
oldest and most popular dyes [1]. It is as a natural dye, originally
extracted from plants such as Indigofera tinctoria [2]. Novel chem-
ical and biological routes for the synthesis of indigo were sought as
the demand for indigo started to increase. For example, Baeyer-
Drewson synthesis is the most well-known organic reaction used
for indigo production [3]. In that synthetic route, 2-
nitrobenzaldehyde and acetone undergo an aldol condensation
reaction, followed by cyclization and oxidative dimerization to
produce indigo. This synthetic method was once one of the most
useful routes to produce indigo. However, the Baeyer-Drewson
synthesis was a relatively impractical process for the synthesis of
indigo at the industrial-scale, and was eventually replaced by the
preparation of indigo from aniline. Aniline is readily obtained and
can be used to synthesize N-(2-carboxyphenyl)glycine, an indigo
precursor [4]. The production of indigo from N-(2-carboxyphenyl)
* Corresponding author.
E-mail address: kychoi@ajou.ac.kr (K.-Y. Choi).
http://dx.doi.org/10.1016/j.dyepig.2017.01.029
0143-7208/© 2017 Elsevier Ltd. All rights reserved.

30
H.-J. Kim et al. / Dyes and Pigments 140 (2017) 29e35
FMO-dependent indigo production has shown that Escherichia coli
(E. coli) that recombinantly produce FMO from Corynebacterium
glutamicum can produce 685 mg/L of indigo and 103 mg/L of
were measured following reduction by sodium dithionite, via UV/
vis spectrophotometry (scanning wavelengths from 400 to 500 nm)
[22]. Briefly, binding spectra were recorded following reduction of
CYP102A_scat protein (oxidized form) by sodium dithionite
(reduced form), followed in turn by the bubbling of carbon mon-
oxide gas into the enzyme solution (CO-bound form). The protein
concentration was subsequently estimated using reduced CO versus
reduced difference spectra. Using an extinction coefficient of
91.9 mMꢁ1 cmꢁ1, at a wavelength of 450 nm, the recombinantly
produced protein content was determined [22,23].
indirubin from 2.5 g/L of L-tryptophan [15].
Here, we demonstrate biosynthesis of indigo dye from glucose
using a recombinant E. coli strain producing a cytochrome P450
monooxygenase (CYP). CYP enzymes are heme-thiolate proteins,
which catalyze the introduction of one oxygen atom from molec-
ular oxygen to a target substrate molecule in a regioselective and
stereoselective manner [16e18]. A few examples of CYP-dependent
production of indigo dye have been reported. Some CYP102A1 BM3
mutants were generated through rational design and site-
saturation mutagenesis for indole hydroxylation activity [9]. A
colorimetric colony method, based on the blue pigment generated
from indigo, was utilized as a high-throughput screening assay to
find active variants with enzymatic activity towards aromatic
compounds [19]. Until now, indigo production using native CYP
enzymes in recombinant microbial hosts has never been reported.
A few attempts have been made using mutant CYPs [20,21]. In this
report we demonstrate, for the first time, indigo production using a
recombinant E. coli strain producing the wild-type CYP102A_scat
enzyme isolated from Streptomyces cattleya (S. cattleya). Further-
more, increased indigo production was achieved by optimal feeding
2.5. Indigo production using E. coli expressing CYP102A_scat
Cells producing CYP102A_scat were cultured in LB media and
the whole cell reactions were initiated by adding IPTG solution
(final concentration: 12.5
mM) to the media, along with the heme
precursor -aminolevulinic acid (final concentration: 0.25 mM).
d
Whole cell production proceeded at 30 ꢀC for 48 h in a high speed
incubator (200 rpm), after which the reaction was quenched by the
addition of an equal volume of DMSO, followed by vigorous vor-
texing. The mixtures were then centrifuged at 13000 rpm for
10 min, after which the blue-colored DMSO layer containing indigo
was separated. The prepared samples were structurally and quan-
titatively analyzed using GC/MS, 1H NMR spectroscopy, and other
spectroscopic techniques.
of indigo precursors such as glucose, indole, and
L-tryptophan.
2. Materials and methods
2.6. Structural and quantitative analysis of biosynthetically
produced indigo
2.1. Chemical reagents
All chemical reagents used in this study were of analytical grade
or higher. Indigo, indole, glucose, and -tryptophan were purchased
from Sigma-Aldrich Korea (Suwon, South Korea).
The fractions collected from the indigo producing cell culture
were separated by centrifugation and further taken for TLC, HPLC,
and 1H NMR. For TLC analysis, the mobile phase was composed of
chloroform: hexane: methanol (5:4:1). The indigo standard solu-
tion was prepared by dissolving 262 mg of synthetic indigo in 1 mL
L
2.2. Phylogenetic analyses
of DMSO and diluted up to several
digo products were also separated with HPLC equipped with a C18
reverse phase column (Zorbax extend-C18 Waters,
m, Agilent, USA) and eluted at 1.0 mL/min
with ACN/Water (50:50 v/v). The absorbance of the eluent was
monitored at 540 nm.
mM ranges using methanol. In-
Amino acid sequences were aligned using the ClustalW2 pro-
gram via the European Bioinformatics Institute website (http://
www.ebi.ac.uk/Tools/msa/clustalw2/). Alignments were visualized
with Bio-edit software (Fig. 1).
250 mm ꢂ 4.6 mm, 3.5
m
2.3. Heterologous expression of the gene encoding CYP102A_scat in
E. coli
For quantitative analysis of the reaction products, the absorption
intensity of the extracted solution was monitored at 610 nm by UV/
vis spectrometry. The production yield of indigo was further
determined using a standard calibration curve obtained using the
same quantification methods with commercially available syn-
thetic indigo. In addition, the collected indigo product was analyzed
by 1H NMR spectroscopy, and the resulting chemical shifts were
compared to that of synthetic indigo.
The DNA sequence information for CYP102A_scat was obtained
from the open-access website: (http://avermitilis.ls.kitasato-u.ac.
jp/). The encoding gene was amplified from its genome and the
PCR product was cloned into a pET-28a(þ) expression vector
(Novagen, Madison, WI). The pET-28a(þ)-CYP102A_scat plasmid
was subsequently transformed into E. coli BL21(DE3), after which,
the transformants were grown in LB medium containing 50
of kanamycin at 37 ꢀC, until an OD600 of 0.8 was attained. At that
point, isopropyl- -thiogalactopyranoside (IPTG) was added to
obtain a final concentration of 12.5 M, along with 0.25 mM
m
g/ml
3. Results and discussion
b
-D
3.1. Self-sufficient CYP102A_scat and their sequence analysis
m
d-
aminolevulinic acid as a heme precursor, after which the cells were
incubated at 30 ꢀC for 12 h. After induction, 1 mL samples of every
point were collected from the cell culture and prepared for indigo
production analysis. At the same time the cells were subsequently
harvested by centrifugation, washed twice with ice-cold PBS buffer,
resuspended in 50 mM potassium phosphate buffer (pH 7.0) and
used for subsequent CYP102A_scat protein preparation and in-vitro
assay.
Cytochrome P450 (CYP) enzymes are generally classified into
several family, depending on their protein sequence identities. One
of the most studied CYP family is CYP102A family, such as
CYP102A1 BM3, CYP102A2, CYP102A3, CYP102A5, and CYP102A7,
which are reported as long-chain fatty acid hydroxylase [24e28].
Likewise, the novel CYP that we are demonstrating in this manu-
script, CYP102A_scat, also belongs to the CYP102A family and has
very high sequence identities with CYP102A enzymes. As was ex-
pected, CYP102A_scat has several unique motifs, such as a heme
binding domain, a dioxygen binding domain, and a substrate
binding domain (Fig. 1). Among the CYP102A family, CYP102A_scat
has highest sequence identity with CYP102A1 BM3 (41.3%), and
2.4. Spectral features of CYP102A_scat
Absorption spectra of CO-bound CYP102A_scat enzyme samples

H.-J. Kim et al. / Dyes and Pigments 140 (2017) 29e35
31
Fig. 1. Multiple sequence alignment of heme domain and reductase domain of the self-sufficient CYP, CYP102A_scat, with other CYP102A family members. (A) Key amino acid
residues in the CYP102A_scat substrate binding pocket and that affect the opening of the substrate access channel; (B) the mechanistically important I-helix region; and (C) the
heme-binding region. Distinctive differences in the active sites are indicated with red boxes. (For interpretation of the references to colour in this figure legend, the reader is referred
to the web version of this article.)
lowest identity with CYP102A5 (39.1%). Since CYP102A enzymes
exhibit very narrow substrate specificities for long-chain fatty
acids, CYP102A_scat was also expected to have fatty acid hydrox-
ylation activity. Consequently, an attempt was made to characterize
the enzymatic activity of CYP102A_scat following its heterologous
production in E. coli BL21(DE3).
dotted line). In the initial stages of CYP102A_scat enzyme charac-
terization, the media changed color, becoming blue when
CYP102A_scat was produced (Fig. 2B). At this stage, we did not
realize that the color arose from indigo dye. After several repeated
rounds of careful CYP102A_scat production, ensuring definite
autoclave sterilization, it was finally proved that the blue pigment
arose from the heterologous CYP102A_scat production, not from
any contamination source. The unknown pigment was finally
identified upon extraction of the blue pigments from the growth
media, followed by structural analyses using 1H NMR spectroscopy.
3.2. Preparation of recombinant E. coli BL21(DE3) producing a self-
sufficient CYP102A_scat
Recombinant E. coli cells capable of expressing the gene
encoding CYP102A_scat were prepared as demonstrated in mate-
rials and method section. There was a good yield of protein. Re-
combinant CYP102A_scat has an apparent mass of 101.2 kDa,
according to an SDS-PAGE analysis (Fig. 2A). The presence of an
activated CYP fraction was confirmed by a CO (99.99%) binding
assay; CYP102A_scat was reduced by sodium dithionite, and after
that, the sample had CO gas bubbled through it. Scanning the ab-
sorption wavelength from 400 to 550 nm in a UV/vis spectropho-
tometer revealed that the highest absorbance peak occurred at
450 nm, which is a characteristic feature of CYP enzymes (Fig. 2C,
3.3. Structural identification of the unknown blue pigment
The unknown blue pigment was extracted from 1 mL of growth
medium using an equal volume of DMSO. After 10 min of centri-
fugation, the deep blue supernatant was separated and prepared
for analysis by TLC, HPLC, and 1H NMR spectroscopy. A verified
sample of synthetic indigo was purchased from Sigma-Aldrich
Korea and analyzed alongside the unknown pigment, to aid in the
precise analysis of its structure. The product separated by TLC
reveled a single indigo product while indigo standard has two spots

32
H.-J. Kim et al. / Dyes and Pigments 140 (2017) 29e35
Fig. 2. Production of functional CYP102A_scat. (A) SDS-PAGE results; M: marker, T: total proteins, S: proteins in the soluble fraction, I: proteins in the insoluble fraction. (B)
Formation of indigo dye, following production of CYP102A_scat, adding various concentrations of certain intermediates involved in tryptophan synthesis. (C) Oxidized, reduced, and
CO-bound spectral features of CYP102A_scat.
of indigo and indirubin in Fig. 4A. The single spot exactly matched
with the blue indigo spot separated from indigo standard. Also,
HPLC separation of produced blue pigment was attempted and
resulted in a single peak with same retention time of corresponding
standard indigo as well (Fig. 4A). 1H NMR spectroscopic analysis
was attempted to further the detailed identification. The assign-
ment of 1H NMR spectrum for blue pigment is shown in Fig. 4B and
(~1.0 g/L) was recorded around 21 h following the induction by IPTG
(Fig. 5). The glucose concentration-dependence of indigo produc-
tion was investigated, in order to examine the effects of providing
an additional carbon source. Media containing three different
concentrations of glucose were examined for indigo production.
Unanticipated production profiles resulted. The higher the con-
centration of glucose added, the lower was the production of indigo
(Fig. 5). The increased flux from glucose did not improve indigo
production at all, rather it led to a decreased production.
E. coli cells synthesize the amino acid tryptophan through the
shikimate pathway. Glucose is converted into shikimate, then
indole, and finally into tryptophan, by the action of several enzymes
(Fig. 3). The most important intermediate in the synthesis of
tryptophan is indole, which is an intercellular signaling molecule.
Indole has been reported to regulate certain physiological processes
of cells, such as sporulation, biofilm formation, plasmid stability,
and virulence. In order to investigate the dependency of indigo
production on the extracellular feeding of indole, different con-
centrations of indole were added to the growth media and the
resulting indigo production was monitored over time. Interestingly,
the higher the indole concentration in the media, the higher the
the 1H NMR spectrum gave peaks at
d
6.551 ppm,
7.496 ppm corresponding to aromatic ring protons, while at
10.465 ppm corresponding to aromatic C-NH. The chemical shifts
d 6.990 ppm, and
d
d
observed were well-matched with those of the reference, synthetic
indigo. In GC/MS analysis, a 262 m/z reading for the isolated blue
compound demonstrated that its molecular weight matched that of
the verified, synthetic indigo (data not shown). Using these struc-
tural techniques, the blue pigment generated by heterologous
production of CYP102A_scat was finally identified as indigo dye.
3.4. Optimization of indigo production by adding synthetic
precursors of indigo
In order to quantify the production of indigo by the cells pro-
ducing CYP102A_scat, the time-dependence of indigo production
was monitored in LB medium. The highest indigo production
indigo production that was achieved. With the provision of 20
mM
of additional indole, the indigo production reached 2.0 0.1 g/L,

H.-J. Kim et al. / Dyes and Pigments 140 (2017) 29e35
33
Fig. 3. Synthetic pathways of indigo production in E. coli cells. Cells utilize the shikimate pathway for indole and tryptophan synthesis. Intracellular indole becomes intercepted by
heterologously produced CYP102A_scat, which catalyzes C-5 specific hydroxylation of indole, resulting in indigo production.
Fig. 4. Separation and structural analysis of produced indigo. (A) Separation of generated indigo by TLC and HPLC. (B) Structural analysis of generated indigo by 1H NMR analysis.

34
H.-J. Kim et al. / Dyes and Pigments 140 (2017) 29e35
1.6 0.3 g/L with the addition of 0.2 mM of
Finally, the combinatorial effects from both of indole and
tryptophan supplementation was investigated by varying concen-
tration of indole while the -tryptophan concentration being con-
stant for 30 h. The optimum Indigo production reached up to
3.8 0.1 g/L by adding 20 M of extracellular indole and 0.2 mM of
-tryptophan to the LB media at 25 h after IPTG induction (Fig. 6C).
L-tryptophan (Fig. 6B).
L-
L
m
L
And the production rate of indigo was measured as 0.15 g/L/hr with
the same optimized condition. To our knowledge, this is a record
and the highest yield achieved so far.
4. Conclusions
◊
Fig. 5. Indigo production over 29 h in LB medium ( line, without additional glucose)
and the effect on indigo production of adding additional glucose to the LB medium (B;
20 mg/L, -; 100 mg/L, C; 200 mg/L).
Here we have isolated a unique self-sufficient monooxygenase
from S. cattleya, termed ‘CYP102A_scat’. The gene encoding
CYP102A_scat was cloned, and then expressed in E. coli BL21(DE3),
in order to investigate its characteristic features. The amino acid
sequence of CYP102A_scat is highly similar with other CYP102A
family members. Unlike other CYP102A species, CYP102A_scat
uniquely produces a blue-dye following IPTG induction in LB me-
dium. The isolated blue pigment (1.0 g/L) was eventually identified
as indigo using TLC, HPLC separation, and 1H NMR spectroscopic
analysis. Additional feeding of glucose did not increase the pro-
ductive yield of indigo. The highest productive yield of indigo was
which amounts to a doubling of the production yield (Fig. 6A).
Along with indole supplementation, the effect of additional L-
tryptophan in the LB media on the production of indigo was also
investigated and resulted in increased indigo production up to
achieved by adding both 20 mM of indole and 0.2 mM of L-trypto-
phan to the culture medium, which boosted indigo production to
3.8 g/L. This novel, self-sufficient CYP102A enzyme, is the first self-
sufficient CYP that exhibits indole hydroxylation activity, resulting
in indigo productiondwithout having to mutate the wild-type
enzyme. Further investigation of
a CYP102A_scat structural
model, together with indole docking simulations extends our un-
derstanding of the indole hydroxylation activity, and these insights
could be applied to other self-sufficient CYP enzymes in the future.
Acknowledgements
The authors have declared no conflict of interest. This work was
supported by the National Research Foundation of Korea (NRF)
grant funded by the Korea government (MEST) (NRF-
2015R1A2A2A04006014, NRF-2015M1A5A1037196). And this work
was also supported by Industrial Strategic Technology Develop-
ment program (No.10044604), funded by the Ministry of Trade,
Industry and Energy (MOTIE/KEIT, Korea).
References
[1] Stasiak N, Kukula-Koch W, Glowniak K. Modern industrial and pharmaco-
logical applications of indigo dye and its derivativesea review. Acta Pol Pharm
2014;71(2):215e21.
[2] Sarangi BK, Minami Y, Thul ST. RNA-Seq analysis for indigo biosynthesis
pathway genes in Indigofera tinctoria and Polygonum tinctorium. Genom
Data 2015;6:212e3.
[3] Drewson ABaV. Darstellung von Indigblau aus Orthonitrobenzaldehyd.
Berichte Dtsch Chem Ges 1882;15(2):2856e64.
[4] Bailey GC. RSP. NOTES-synthesis of indigo from fumaric acid and aniline;
preparation of dibrom-succinic acid; fusion of dianilido-succinic acid to in-
digo. J Am Chem Soc 1922;44(1):215e6.
[5] Ferber KH. Toxicology of indigo. A review. J Environ Pathol Toxicol Oncol
1987;7(4):73e83.
[6] Butterworth KR, Hoosen J, Gaunt IF, Kiss IS, Grasso P. Long-term toxicity of
indigo carmine in mice. Food Cosmet Toxicol 1975;13(2):167e76.
[7] Chia MA, Musa RI. Effect of indigo dye effluent on the growth, biomass pro-
duction and phenotypic plasticity of Scenedesmus quadricauda (Chlor-
ococcales). An Acad Bras Cienc 2014;86(1):419e28.
[8] Vuorema A, John P, Keskitalo M, Mahon MF, Kulandainathan MA, Marken F.
Anthraquinone catalysis in the glucose-driven reduction of indigo to leuco-
indigo. Phys Chem Chem Phys 2009;11(11):1816e24.
Fig. 6. (A) Indigo production with various concentrations of supplemental indole in
the LB medium. ( ; 0 mM, B; 1 mM, -; 10 mM, C; 20 mM), (B) Indigo production with
various concentration of supplemental L-tryptophan in the LB medium ( ; 0 mM, B;
0.01 mM, -; 0.1 mM, C; 0.2 mM), (C) Indigo production with various concentration of
supplemental indole and 0.2 mM of L-tryptophan in the LB medium ( ; 0 mM, B;
0.01 mM, -; 0.1 mM, C; 0.2 mM of indole).
◊
◊
[9] Lu Y, Mei L. Co-expression of P450 BM3 and glucose dehydrogenase by re-
combinant Escherichia coli and its application in an NADPH-dependent indigo
production system. J Ind Microbiol Biotechnol 2007;34(3):247e53.
◊

H.-J. Kim et al. / Dyes and Pigments 140 (2017) 29e35
35
[10] Qu Y, Zhang X, Ma Q, Deng J, Deng Y, Van Nostrand JD, et al. Microbial
community dynamics and activity link to indigo production from indole in
bioaugmented activated sludge systems. PLoS One 2015;10(9):e0138455.
[11] Shi S, Ma F, Sun T, Li A, Zhou J, Qu Y. Biotransformation of indole to indigo by
the whole cells of phenol hydroxylase engineered strain in biphasic systems.
Appl Biochem Biotechnol 2013;169(4):1088e97.
[19] Park SH, Kim DH, Kim D, Kim DH, Jung HC, Pan JG, et al. Engineering bacterial
cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme
activity using indigo formation. Drug Metab Dispos 2010;38(5):732e9.
[20] Pengpai Z, Sheng H, Lehe M, Yinlin L, Zhihua J, Guixiang H. Improving the
activity of cytochrome P450 BM-3 catalyzing indole hydroxylation by directed
evolution. Appl Biochem Biotechnol 2013;171(1):93e103.
[12] Doukyu N, Nakano T, Okuyama Y, Aono R. Isolation of an Acinetobacter sp. ST-
550 which produces a high level of indigo in a water-organic solvent two-
phase system containing high levels of indole. Appl Microbiol Biotechnol
2002;58(4):543e6.
[21] Appel D, Lutz-Wahl S, Fischer P, Schwaneberg U, Schmid RD. A P450 BM-3
mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes.
J Biotechnol 2001;88(2):167e71.
[22] Omura T, Sato R.
A new cytochrome in liver microsomes. J Biol Chem
[13] Choi HS, Kim JK, Cho EH, Kim YC, Kim JI, Kim SW. A novel flavin-containing
monooxygenase from Methylophaga sp strain SK1 and its indigo synthesis
in Escherichia coli. Biochem Biophys Res Commun 2003;306(4):930e6.
[14] Lin GH, Chen HP, Huang JH, Liu TT, Lin TK, Wang SJ, et al. Identification and
characterization of an indigo-producing oxygenase involved in indole 3-acetic
acid utilization by Acinetobacter baumannii. Ant Van Leeuwenhoek
2012;101(4):881e90.
1962;237:1375e6.
[23] Koley AP, Buters JT, Robinson RC, Markowitz A, Friedman FK. CO binding ki-
netics of human cytochrome P450 3A4. Specific interaction of substrates with
kinetically distinguishable conformers. J Biol Chem 1995;270(10):5014e8.
[24] Budde M, Maurer SC, Schmid RD, Urlacher VB. Cloning, expression and
characterisation of CYP102A2, a self-sufficient P450 monooxygenase from
Bacillus subtilis. Appl Microbiol Biotechnol 2004;66(2):180e6.
[15] Ameria SP, Jung HS, Kim HS, Han SS, Kim HS, Lee JH. Characterization of a
flavin-containing monooxygenase from Corynebacterium glutamicum and its
application to production of indigo and indirubin. Biotechnol Lett 2015;37(8):
1637e44.
[16] McDonnell AM, Dang CH. Basic review of the cytochrome p450 system. J Adv
Pract Oncol 2013;4(4):263e8.
[17] Girvan HM, Munro AW. Applications of microbial cytochrome P450 enzymes
in biotechnology and synthetic biology. Curr Opin Chem Biol 2016;31:
136e45.
[25] Chowdhary PK, Alemseghed M, Haines DC. Cloning, expression and charac-
terization of a fast self-sufficient P450: CYP102A5 from Bacillus cereus. Arch
Biochem Biophys 2007;468(1):32e43.
[26] Dietrich M, Eiben S, Asta C, Do TA, Pleiss J, Urlacher VB. Cloning, expression
and characterisation of CYP102A7, a self-sufficient P450 monooxygenase from
Bacillus licheniformis. Appl Microbiol Biotechnol 2008;79(6):931e40.
[27] Choi KY, Jung E, Jung DH, Pandey BP, Yun H, Park HY, et al. Cloning, expression
and characterization of CYP102D1, a self-sufficient P450 monooxygenase
from Streptomyces avermitilis. FEBS J 2012;279(9):1650e62.
[18] Urlacher VB, Girhard M. Cytochrome P450 monooxygenases: an update on
perspectives for synthetic application. Trends Biotechnol 2012;30(1):26e36.
[28] De Mot R, Parret AH. A novel class of self-sufficient cytochrome P450 mon-
ooxygenases in prokaryotes. Trends Microbiol 2002;10(11):502e8.