Production and anti-melanoma activity of methoxyisoflavones from the biotransformation of genistein by two recombinant Escherichia coli strains

Article abstract of DOI:10.3390/molecules22010087

Biotransformation of the soy isoflavone genistein by sequential 3′-hydroxylation using recombinant Escherichia coli expressing tyrosinase from Bacillus megaterium and then methylation using another recombinant E. coli expressing O-methyltransferase from S

Full text of DOI:10.3390/molecules22010087

molecules
Communication
Production and Anti-Melanoma Activity
of Methoxyisoflavones from the
Biotransformation of Genistein by
Two Recombinant Escherichia coli Strains
Chien-Min Chiang 1, Yu-Jhe Chang 2, Jiumn-Yih Wu 3,4,* and Te-Sheng Chang 2,
*
1
Department of Biotechnology, Chia Nan University of Pharmacy and Science, No. 60, Sec. 1,
Erh-Jen Rd., Jen-Te District, Tainan 71710, Taiwan; cmchiang@mail.cnu.edu.tw
Department of Biological Sciences and Technology, National University of Tainan, No. 33, Sec. 2,
Shu-Lin St., Tainan 70005, Taiwan; roger199382@yahoo.com.tw
Institute of Biotechnology and Chemical Engineering, I-Shou University, No. 1, Sec. 1, Syuecheng Rd.,
Dashu District, Kaohsiung 84001, Taiwan
2
3
4
Center for General Education, National Quemoy University, No. 1, University Road., Jin-Ning Township,
Kinmen County 892, Taiwan
*
Correspondence: wujy@nqu.edu.tw (J.-Y.W.); mozyme2001@gmail.com (T.-S.C.);
Tel.: +886-82-313310 (J.-Y.W.); Tel./Fax: +886-6-2606153 (T.-S.C.); Fax: +886-82-313797 (J.-Y.W.)
Academic Editor: David J. Newman
Received: 17 November 2016; Accepted: 3 January 2017; Published: 4 January 2017
Abstract: Biotransformation of the soy isoflavone genistein by sequential 30-hydroxylation using
recombinant Escherichia coli expressing tyrosinase from Bacillus megaterium and then methylation using
another recombinant E. coli expressing O-methyltransferase from Streptomyces peucetius was conducted.
The 0results showed that two metabolites were0produced from the biotransformation, identified as
0
0
5,7,4 -trihydroxy-3 -methoxyisoflavone and 5,7,3 -trihydroxy-4 -methoxyisoflavone, respectively, based
on their mass and nuclear magnetic resonance spectral data. 5,7,40-Trihydroxy-30-methoxyisoflavone
showed potent antiproliferative activity toward mouse B16 melanoma cells with an IC50 value of
68.8
µM. In contrast, the compound did not show any cytotoxicity toward mouse normal fibroblast cells,
even at 350 µM concentration. The results of the present study offer insight on the production of both
0
0
0
0
5,7,4 -trihydroxy-3 -methoxyisoflavone and 5,7,3 -trihydroxy-4 -methoxyisoflavone by two recombinant
E. coli strains and the potential anti-melanoma applications of 5,7,40-trihydroxy-30-methoxyisoflavone.
Keywords: genistein; melanoma; methoxyisoflavone; methyltransferase; tyrosinase
1. Introduction
Isoflavones are a kind of flavonoid widely distributed in several plants, such as red clover and
soybeans [
These two isoflavones have received much attention over the past few decades due to their possible
roles in preventing certain hormone-dependent diseases [ ]. In recent years, modification of soy
1,2]. Genistein and daidzein are the two major isoflavone aglycones found in soybeans.
3
isoflavones using gene-engineered microorganisms has also been of interest, as the bioactivity of
compounds with different structures is dramatically altered [4].
Hydroxylation and methylation are two common modifications of flavonoids occurring in Nature.
Both modifications produce more complex flavonoids, which sometimes possess higher bioactivity
than their precursors. For conducting such modifications in laboratories, researchers have constructed
several gene-engineered microorganisms. Using genetically-modified microorganisms to perform
biotransformations has the advantage of allowing the rational design of desirable biotransformation
Molecules 2017, 22, 87; doi:10.3390/molecules22010087
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products. Moreover, some microorganisms, such as the bacterium E. coli or the yeast Pichia pastoris,
are easily handled in both biotransformation and scale-up processes [ ]. Therefore, using genetically
5
modified microorganisms to carry out biotransformations of isoflavones is an interesting issue.
Recently, Lee et al. used a recombinant E. coli expressing tyrosinase from Bacillus megaterium to
catalyze the ortho-hydroxylation of daidzein and genistein in the presence of borate and ascorbic acid [
The biotransformation system was also successfully applied to catalyze the ortho-hydroxylation of the
soy isoflavone glycosides daidzin and genistin [ ]. On the other hand, a recombinant E. coli expressing
O-methyltransferase from Streptomyces peucetius was demonstrated to catalyze the O-methylation
of ortho-dihydroxyisoflavone [ ]. Based on the results of these previous studies, it is possible to
6].
7
8
use the two recombinant E. coli strains to conduct dual modifications, including hydroxylation and
O-methylation, on soy isoflavones. In the present study, biotransformation of the soy isoflavone
genistein, first by 30-hydroxylation using the recombinant E. coli expressing B. megaterium tyrosinase
and second by O-methylation using the recombinant E. coli expressing S. peucetius O-methyltransferase,
was performed, and the anti-proliferative activity of the resulting biotransformation metabolites on
both mouse melanoma and normal fibroblast cells was also determined.
2. Results
2.1. Biotransformation of Genistein by the Two Recombinant E. coli Strains and Purification and Identification
of the Biotransformation Products
In the present study, commercial genistein soy isoflavone was used as a biotransformation
precursor, which appeared at retention time of 8.34 min in the ultra-performance liquid chromatography
(UPLC) analysis (Figure 1a). Figure 1b shows the UPLC profile of the biotransformation product of
genistein using recombinant E. coli expressing B. megaterium tyrosinase in the presence of 500 mM of
borate (pH 9.0) and 10 mM of ascorbate. As shown in the figure, the genistein peak almost completely
disappeared and a new peak appeared at a retention time of 6.42 min. According to the results by Lee
et al. [6] and our previous study [7
], the new peak at 6.42 min should be 30-hydroxygenistein. In the
biotransformation, genistein was almost completed converted. The result is consistent with that of
Lee et al., who showed that a 93% conversion yield was achieved using the recombinant E. coli for
30-hydroxylation of genistein [6].
0
In a second biotransformation, the produced 3 -hydroxygenistein from the first biotransformation
reaction was incubated with the recombinant E. coli expressing S. peucetius O-methyltransferase in
the presence of 1 mM of S-adenosylmethionine (SAM) for 24 h. Figure 1c shows the UPLC profile of
the second biotransformation reaction product. In the figure, two new peaks appear—compounds (1)
and (2), with retention times of 8.52 and 8.81 min, respectively. The results revealed that the
30-hydroxygenistein produced by the first biotransformation reaction was further biotransformed in
the second biotransformation reaction.
The metabolites of the second reaction were isolated using a preparative high-performance liquid
chromatography (HPLC) method and were identified using spectrophotometric methods. Compound
1
showed an [M + H]+ ion peak at m/z: 301 in the electrospray ionization mass (ESI-MS) spectrum,
corresponding to the molecular formula C16H12O6. Then both 1H and 13C nuclear magnetic resonance
(NMR) experiments including heteronuclear multiple quantum coherence (HMQC), heteronuclear
multiple bond connectivity (HMBC), nuclear Overhauser effect spectroscopy (NOESY), and correlation
spectroscopy (COSY) spectra were performed and the 1H- and 13C-NMR signal assignments were
conducted accordingly. The HMBC spectrum revealed a methoxyl proton signal at
to carbon resonance at
147.3 (C-30), and the NOESY spectrum revealed the distance proximity
between protons at
7.13 (d, H-20). Based on these spectral data and
δ 3.79(s) correlated
δ
δ
3.79(s) and protons at δ
comparison with the 1H-NMR and 13C-NMR data in the literature [
as 5,7,40-trihydroxy-30-methoxyisoflavone.
9], compound 1 was characterized

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0.14
0.12
0.1
Genistein
0.08
0.06
0.04
0.02
0
0
2
4
6
8
10
Retention Time (min)
(a)
0.025
3'-Hydroxygenistein
0.02
0.015
0.01
0.005
0
0
2
4
6
8
10
Retention Time (min)
(b)
0.02
0.015
0.01
Compound (1)
Compound (2)
0.005
0
0
1
2
3
4
5
6
7
8
9
10
Retention Time (min)
(c)
Figure 1. UPLC profiles of the biotransformation precursor genistein (a); the products of the first
biotransformation reaction using the recombinant E. coli expressing tyrosinase (
b
); the products of the
second biotransformation reaction using the recombinant E. coli expressing O-methyltransferase (c).
The detailed protocols for biotransformation and UPLC are described in Materials and Methods.
Compound
methoxyl proton signal at
spectrum revealed the distance proximity between protons at
2
showed an [M + H]+ ion peak at m/z: 301. The HMBC spectrum revealed a
0
δ
3.79(s) correlated to the carbon resonance at
δ
157.4 (C-4 ), and the NOESY
0
δ
3.79(s) and at
δ
7.02 (d, H-5 ). Based on
these spectral data and a comparison of the 1H-NMR and 13C-NMR data with literature values [10],
compound
was characterized as 5,7,30-trihydroxy-40-methoxyisoflavone. These results—together
with our previous study [ ] and the study performed by Lee et al. [ ]—reveal that genistein
2
7
,8
6

Molecules 2017, 22, 87
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was first converted into 30-hydroxygenistein by the recombinant E. coli expressing B. megaterium
tyrosinase, and then the resulting 30-hydroxygenistein was further transformed by recombinant
E. coli expressing S. peucetius O-methyltransferase into 5,7,40-trihydroxy-30-methoxyisoflavone or
5,7,30-trihydroxy-40-methoxyisoflavone. Scheme 1 shows a diagram of the biotransformation of
genistein by the two recombinant E. coli strains.
HO
O
Recombinant E. coli expressing
B. megaterium tyrosinase
HO
O
3'
4'
OH
OH
3'
4'
Borate/Ascorbic acid
OH
O
OH
OH
O
Genistein
3'-Hydroxygenistein
(a)
HO
O
OMe
3'
4'
OH
OH
O
HO
O
Recombinant E. coli expressing
S. peucetius O-methyltransferase
5,7,4'-Trihydroxy-3’-
methoxyisoflavone
OH
OH
3'
4'
SAM
OH
O
HO
O
OH
3'-Hydroxygenistein
3'
4'
OMe
OH
O
5,7,3'-Trihydroxy-4'-
methoxyisoflavone
(b)
Scheme 1. Diagram of the biotransformation of genistein by the two recombinant E. coli.
0
(
a
) Genistein was firstly biotransformed to 3 -hydroxygenistein by recombinant E. coli that expressed
B. megaterium tyrosinase; (b
) The produced 30-hydroxygenistein was then biotransformed to
5,7,40-trihydroxy-30-methoxyisoflavone and 5,7,30-trihydroxy-40-methoxyisoflavone by recombinant
E. coli that expressed S. peucetius O-methyltransferase.
2.2. Anti-Melanoma Activity of the Biotransformation Products
The search for new melanogenesis inhibitors for use in skin-whitening cosmetics is an interesting
topic [8,
11
,12]. In the assay for evaluating melanogenesis inhibitory activity, mouse B16 melanoma cells
are used [12
,
13]. After producing the two methoxyisoflavones in the present study, the melanogenesis
inhibitory activity of the two methoxyisoflavones were determined by culturing B16 melanoma
cells with them. To our surprise, the two methoxyisoflavones were found to be highly toxic to the
melanoma cells. Table 1 shows the cytotoxicity experiment results. Among the tested compounds,
5,7,40-trihydroxy-30-methoxyisoflavone exhibited the most potent cytotoxicity toward the B16
melanoma cells, with an IC50 value of 68.1
µM. To evaluate the selectivity of the anti-melanoma activity,
the cytotoxicity experiments were performed again using normal mouse fibroblast cells. The results
0
0
showed that 5,7,4 -trihydroxy-3 -methoxyisoflavone exhibited no significant cytotoxicity toward mouse
normal fibroblast cells, even at 350 M concentration (Table 1). In addition, the biotransformation
µ

Molecules 2017, 22, 87
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precursor genistein also showed potent cytotoxicity toward B16 melanoma cells, with an IC50 value of
70.3 µM, but little cytotoxicity toward mouse normal fibroblast cells (Table 1).
Table 1. Cell survival 1 of the studied isoflavones toward mouse B16 melanoma cells and mouse normal
fibroblast cells.
Cells
Melanoma
1
Fibroblast
1
Isoflavone
Genistein
2
Genistein
2
Control
11 µM
22 µM
44 µM
88 µM
175 µM
350 µM
100 ± 3.21
102.41 ± 4.52
77.55 ± 3.55 *
55.28 ± 2.99 *
44.41 ± 1.92 *
41.72 ± 3.11 *
39.81 ± 1.91 *
70.3
100 ± 4.48
99.15 ± 7.42
80.77 ± 4.53 *
57.9 ± 5.18 *
40.38 ± 1.69 *
32.05 ± 0.9 *
33.54 ± 2.89 *
68.1
100 ± 6.64
92.67 ± 2.27
83.57 ± 4.43 *
82.79 ± 3.39 *
81.72 ± 2.78 *
74.19 ± 0.29 *
53.86 ± 3.00 *
420.3
100 ± 4.12
97.81 ± 4.67
102.33 ± 3.76
98.91 ± 3.41
96.7 ± 2.89
92.11 ± 3.12
85.77 ± 1.98 *
N.D. 3
100 ± 3.41
103.68 ± 3.57
102.09 ± 3.86
94.69 ± 3.71
97.69 ± 2.28
102.09 ± 3.86
89.21 ± 4.99
N.D.
100 ± 0.22
102.42 ± 4.97
99.28 ± 0.42
93.58 ± 2.43 *
89.77 ± 1.18 *
84.26 ± 1.16 *
71.70 ± 1.00 *
N.D.
2
IC50 (µM)
1
The mean (n = 3) is shown, and the S.D. is represented by error. A value of p < 0.05 (*) from a student’s
t-test analysis by comparing the data with that of the control was considered to be statistically significant.
2 The IC50 values represent the concentrations required for 50% inhibition on the cell growth. 3 N.D. represented
not determined.
3. Discussion
The present study successfully developed a two-step biotransformation process for the production
of 5,7,40-trihydroxy-30-methoxyisoflavone ( ) and 5,7,30-trihydroxy-40-methoxyisoflavone (
) first
1
2
using recombinant E. coli expressing B. megaterium tyrosinase and then using recombinant E. coli
expressing S. peucetius O-methyltransferase. To our knowledge, this is the first report of the
bio-production of the two methoxyisoflavones. Although 5,7,40-trihydroxy-30-methoxyisoflavone
and 5,7,30-trihydroxy-40-methoxyisoflavone have been isolated from several plant parts, such as the
stems of Erycibe expansa [14] and the branch wood of Andira surinamensis [10], they are rare in Nature.
Development of an easy method for producing the two methoxyisoflavones would open the research
field to exploring the bioactivity of these two compounds in the future. In addition, we found
that 5,7,40-trihydroxy-30-methoxyisoflavone exhibited potent anti-proliferative activity on mouse B16
melanoma cells, with an IC50 value of 68.8
µM, but showed no significant growth inhibition on mouse
normal fibroblast cells, even at 350
µ
M concentration (Table 1). The preliminary findings highlight the
potential use of 5,7,40-tr0ihydroxy-30-methoxyisoflavone for its anti-melanoma activity.
0
In addition to 5,7,4 -trihydroxy-3 -methoxyisoflavone, in the present study the biotransformation
precursor genistein was also found to display potent cytotoxicity against B16 melanoma cells
(Table 1). The result was consistent with that reported by Record et al. [15]. In fact, genistein
also shows potent cytotoxicity to other melanoma cells [16]. Although genistein possesses potent
anti-melanoma activities, however, it suffers from the apparent drawback of low bioavailability [17].
It has been reported that methylation of free hydroxyl groups in flavonoids dramatically increases
their metabolic stability and enhances cell membrane transport, leading to facilitated absorption,
and this greatly increases oral bioavailability [18,19]. Therefore, the methoxygenistein derivative
5,7,40-trihydroxy-30-methoxyisoflavone produced in the present study may possess more potential in
cancer therapy than its precursor genistein. However, more detailed studies need to be done in the
future to confirm this.
Regarding substrate specificity, both enzymes used in the present study possess a broad
substrate spectrum. In addition to the natural substrates tyrosine and L-3,4-dihydroxyphenylalanine
(L-DOPA), B. megaterium tyrosinase has been proven to transform the isoflavone aglycones daidzein
and genistein, the isoflavone glycosides daidzin and genistin, and the stilbene resveratrol [6,7].
The range of the chemical structures of the catalyzed substrates is quite large and includes
a single benzene ring (tyrosine and L-DOPA), two benzene rings (resveratrol), or three rings
(flavonoids). S. peucetius O-methyltransferase belongs to the class I O-methyltransferases (also known

Molecules 2017, 22, 87
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as caffeoyl coenzyme A OMTs or CCoAOMTs), which are involved in methylation of phenolic
hydroxyl residues [20]. The enzyme has been shown to catalyze many flavonoids, including
flavonols (quercetin, rutin), flavones (7,8-dihydroxyflavone, luteolin), a flavanone (naringenin),
and isoflavonoids (daidzein, 8-hydroxydaidzein, and formononetin) [8,19]. Based on the results of the
previous studies, it is highly possible that the two-step biotransformation process developed in the
present study could also be applied to other flavonoids or even simple phenolic compounds. Currently
several simple phenolic or flavonoid precursors, including daidzin, genistin, daidzein, resveratrol,
apigenin, naringin, naringenin, morin, arbutin, hordenine, and synephrine, are being tested by the
two-step biotransformation system in our laboratory. Through the two step biotransformation process
developed in the present study, many methoxyflavonoids derivatives could be produced in the future.
In addition to the actual production of novel methoxyflavonoids, research on the multiple bioactivities
of the produced methoxyflavonoids would also be an interesting future area of study.
4. Materials and Methods
4.1. Microorganisms, Animal Cells, and Chemicals
Both recombinant E. coli expressing B. megaterium tyrosinase and recombinant E. coli expressing
S. peucetius O-methyltransferase were constructed in our laboratory per our previous work [7,8].
We purchased both mouse B16-F10 melanoma cells BCRC 60031 and mouse normal fibroblast cells
BCRC 60203 from the Bioresources Collection and Research Center (BCRC, Food Industry Research
and Development Institute, Hsinchu, Taiwan). Genistein, isopropyl-β-D-thiogalactopyranoside
(IPTG), DMSO, SAM, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were
purchased from Sigma (St. Louis, MO, USA). The other reagents and solvents used were of high quality
and were purchased from commercially available sources.
4.2. Preparation of Biocatalyst
The lyophilized recombinant E. coli cells were used as the biocatalyst in this study, and we
prepared these cells based on our previous work [7], as described briefly below. The recombinant
cells were cultured in 100 mL of Luria-Bertani (LB) medium containing 100 µg/mL of ampicillin with
180 rpm shaking at 37 ◦C. As the optical density at 600 nm reached 0.6, 0.1 mM of IPTG was added to
◦
induce gene expression. The cells were continuously cultivated at 18 C for another 24 h. At the end of
the cultivation, the cells were harvested and washed once with phosphate-buffered saline (PBS, pH 6.8).
Cell pellets from the washed cells were lyophilized by a freeze dryer.
4.3. Biotransformation of Both Hydroxylation and Methylation
Genistein (200 mg, 100 mg/mL in DMSO) was added to reaction mixture (100 mL) containing
500 mM of borate pH 9.0, 10 mM of ascorbic acid, and 20 mg of the lyophilized recombinant E. coli
expressing B. megaterium tyrosinase to start the hydroxylation reaction. The reaction was run at 50 ◦C
and 200 rpm for 1.5 h in an incubator. At the end of the reaction, 1 M HCl (20 mL) was added to stop the
reaction. Then, ethyl acetate (120 mL) was added to extract the isoflavones from the reaction mixture.
The ethyl acetate fraction was dried under vacuum. The dried mass (373 mg) was dissolved in DMSO
(100 mg/mL), analyzed by UPLC, and added into another 100 mL of reaction mixture containing 1 mM
of SAM and 20 mg of the lyophilized recombinant E. coli expressing S. peucetius O-methyltransferase
to start the methylation reaction. The reaction was run at 40 ◦C and 200 rpm for 24 h in an incubator.
At the end of the reaction, ethyl acetate (100 mL) was added to extract the isoflavones from the reaction
mixture. The ethyl acetate fraction was dried under vacuum. The dried mass was dissolved in 100 mL
of 50% (v/v) methanol, analyzed by UPLC, and used for purification of metabolites.

Molecules 2017, 22, 87
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4.4. UPLC Analysis
Biotransformation mixtures were analyzed with a UPLC system (Acquity UPLC H-Class, Waters,
Milford, MA, USA) equipped with an analytic C18 reversed-phase column (Acquity UPLC BEH C18,
1.7
µ
m, 2.1 mm i.d.
×
100 mm, Waters). The operation conditions included a gradient elution using
water (A) containing 1% (v/v) acetic acid and methanol (B) with a linear gradient for nine min with
35% to 80% B and for another one minute with 80% to 85% B at a flow rate of 0.3 mL/min, injection
volume of 0.2 µL, and detection of the absorbance at 260 nm.
4.5. Purification and Identification of Biotransformation Products
The final reaction mixture obtained from the above biotransformation (in 100 mL of 50% methanol)
was centrifuged at 10,000 rpm and filtered with a 0.22
Then, the filtrate was injected into a preparative YoungLin HPLC system (YL9100, YL Instrument,
Gyeonggi-do, Korea) equipped with a preparative C18 reversed-phase column (Inertsil, 10 m,
20.0 mm i.d. 250 mm, ODS 3, GL Sciences, Eindhoven, The Netherlands) for purification of
µm nylon membrane under vacuum.
µ
×
the biotransformation products. The operational conditions for the preparative HPLC analysis were
the same as those in the UPLC analysis. The elution corresponding to the peak of the metabolite in
the UPLC analysis was collected, condensed under a vacuum, and then crystallized by freeze drying.
Finally, 27.9 mg of compound (1) and 33.3 mg of compound (2) were obtained. The structures of
the compounds were confirmed with NMR and mass spectral analysis. The mass spectral analysis
was performed on a Finnigan LCQ Duo mass spectrometer (ThermoQuest Corp., San Jose, CA, USA)
equipped with electrospray ionization (ESI). 1H- and 13C-NMR, HMQC, HMBC, COSY, and NOESY
spectra were recorded on an AV-700 NMR spectrometer (Bruker Corp., Billerica, MA, USA) at ambient
temperature. Standard pulse sequences and parameters were used for the NMR experiments, and all
chemical shifts were reported in parts per million (ppm, δ).
5,7,40-Trihydroxy-30-methoxyisoflavone ( ). 1H-NMR (DMSO-d6)
1 δ: 8.34 (1H, s, H-2), 7.13 (1H, d,
J = 2.1 Hz, H-20), 6.98 (1H, d, J = 8.1, 2.1 Hz, H-60), 6.82 (1H, d, J = 8.1, H-50), 6.37 (1H, d, J = 2.2 Hz,
H-8), 6.21 (1H, J = 2.2 Hz, H-6), 3.79 (3H, s, MeO-30); 13C-NMR (DMSO-d6): 180.1 (C-4), 164.8 (C-7),
162.0 (C-5), 157.6 (C-9), 154.1 (C-2), 147.3 (C-30), 146.7 (C-40), 122.3 (C-3), 121.7 (overlap, C-10, C-60),
115.3 (C-50), 113.3 (C-20), 104.3 (C-10), 99.1 (C-6), 93.8 (C-8), 55.7 (OCH3).
0
0
1
5,7,3 -Trihydroxy-4 -methoxyisoflavone (
2). H-NMR (DMSO-d6) δ: 8.28 (1H, s, H-2), 7.02 (1H, d, J = 2.1 Hz,
H-20), 6.96 (1H, d, J = 8.3, H-50), 6.93 (1H, dd, J = 8.3, 2.1 Hz, H-60), 6.32 (1H, d, J = 2.1 Hz, H-8),
6.17 (1H, d, J = 2.1 Hz, H-6), 3.79 (3H, s, MeO-40); 13C-NMR (DMSO-d6)
δ: 179.9 (C-4), 165.8 (C-7),
162.0 (C-9), 157.7 (C-2), 154.0 (C-40), 146.1 (C-30), 123.5 (C-10), 122.0 (C-3), 119.8 (C-60), 116.4 (C-20),
112.0 (C-50), 103.9 (C-10), 99.4 (C-6), 93.9 (C-8), 55.7 (OCH3).
4.6. Determination of Cell Viability
Cell viability was determined as previously reported [8] and described briefly below.
Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) fetal bovine serum was used
◦
to cultivate the tested cells, which were incubated at 37 C in a humidified, CO2-controlled (5%)
incubator. After one day of incubation, the cells were treated with tested drugs for another two days.
The drug-treated cells were then harvested and the cell viability was measured according to our
previous study [8].
5. Conclusions
0
0
The present study first developed a bio-production process for 5,7,4 -trihydroxy-3 -methoxyisoflavone
(1 2), where genistein was firstly biotransformed to
) and 5,7,30-trihydroxy-40-methoxyisoflavone (
30-hydroxygenistein by recombinant E. coli that expressed B. megaterium tyrosinase and the
produced 30-hydroxygenistein was then biotransformed to 5,7,40-trihydroxy-30-methoxyisoflavone

Molecules 2017, 22, 87
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and 5,7,30-trihydroxy-40-methoxyisoflavone by recombinant E. coli that expressed S. peucetius
O-methyltransferase. Moreover, the results of the present study also showed the specific potent
anti-melanoma activity of 5,7,40-trihydroxy-30-methoxyisoflavone in cultured mouse cells.
Acknowledgments: This research was financially supported by grants from the National Scientific Council of
Taiwan (project no. MOST 105-2221-E-024-018-).
Author Contributions: Te-Sheng Chang and Jiumn-Yih Wu conceived and designed the experiments and wrote
the paper; Chien-Min Chiang resolved the chemical structures of compounds (1) and (2). Yu-Jhe Chang performed
the experiments for biotransformation and anti-proliferative activity assay of the tested compounds.
Conflicts of Interest: The authors declare no conflicts of interest.
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Sample Availability: Samples of the compounds 5,7,4 -trihydroxy-3 -methoxyisoflavone and 5,7,3 -trihydroxy-4 -
methoxyisoflavone are available from the authors.
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