Regioselective hydroxylation of trans -resveratrol via inhibition of tyrosinase from streptomyces avermitilis MA4680

Article abstract of DOI:10.1021/cb300222b

Secreted tyrosinase from melanin-forming Streptomyces avermitilis MA4680 was involved in both ortho-hydroxylation and further oxidation of trans-resveratrol, leading to the formation of melanin. This finding was confirmed by constructing deletion mutants of melC₂ and melD ₂ encoding extracellular and intracellular tyrosinase, respectively; the melC2 deletion mutant did not produce piceatannol as well as melanin, whereas the melD2 deletion mutant oxidized resveratrol and synthesized melanin with the same yields, suggesting that MelC2 is responsible for ortho-hydroxylation of resveratrol. Extracellular tyrosinase (MelC2) efficiently converted trans-resveratrol into piceatannol in the presence of either tyrosinase inhibitors or reducing agents such as catechol, NADH, and ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific hydroxylation of trans-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of trans-resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of trans-resveratrol was dependent upon inhibitor concentration. Using 1.8 mg of wild-type Streptomyces avermitilis cells, the conversion yield of 100 μM trans-resveratrol to piceatannol was 78% in 3 h in the presence of 1 mM catechol, indicating 14 μM piceatannol h^-1 DCW mg^-1 specific productivity, which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases.

Full text of DOI:10.1021/cb300222b

Articles
pubs.acs.org/acschemicalbiology
Regioselective Hydroxylation of trans-Resveratrol via Inhibition of
Tyrosinase from Streptomyces avermitilis MA4680
Nahum Lee, Eun Jung Kim, and Byung-Gee Kim*
School of Chemical and Biological Engineering, Institute of Molecular Biology and Genetics, Institute of Bioengineering, Seoul
National University, Seoul, South Korea
S
* Supporting Information
ABSTRACT: Secreted tyrosinase from melanin-forming Streptomyces
avermitilis MA4680 was involved in both ortho-hydroxylation and further
oxidation of trans-resveratrol, leading to the formation of melanin. This
finding was confirmed by constructing deletion mutants of melC2 and melD2
encoding extracellular and intracellular tyrosinase, respectively; the melC2
deletion mutant did not produce piceatannol as well as melanin, whereas the
melD2 deletion mutant oxidized resveratrol and synthesized melanin with
the same yields, suggesting that MelC2 is responsible for ortho-
hydroxylation of resveratrol. Extracellular tyrosinase (MelC2) efficiently
converted trans-resveratrol into piceatannol in the presence of either
tyrosinase inhibitors or reducing agents such as catechol, NADH, and
ascorbic acid. Reducing agents slow down the dioxygenase reaction of tyrosinase. In the presence of catechol, the regio-specific
hydroxylation of trans-resveratrol was successfully performed by whole cell biotransformation, and further oxidation of trans-
resveratrol was efficiently blocked. The yield of this ortho-hydroxylation of trans-resveratrol was dependent upon inhibitor
concentration. Using 1.8 mg of wild-type Streptomyces avermitilis cells, the conversion yield of 100 μM trans-resveratrol to
piceatannol was 78% in 3 h in the presence of 1 mM catechol, indicating 14 μM piceatannol h−1 DCW mg−1 specific productivity,
which was a 14-fold increase in conversion yield compared to that without catechol, which is a remarkably higher reaction rate
than that of P450 bioconversion. This method could be generally applied to biocatalysis of various dioxygenases.
esveratrol is a phytoalexin naturally produced by plants
such as grapes, berries, and peanuts.1−3 A physiological
concentration, S. avermitilis MA4680 was selected. The
supernatant of its whole cell broth degraded tRES more
significantly than the whole cell fraction, indicating that the
enzymes responsible for its oxidation or degradation are
secreted rather than cytoplasmic P450s. Tyrosinase was
identified as the candidate enzyme for the oxidation and
further degradation by sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) and tryptic in-gel digestion
followed by mass spectrometry (MS) analysis, since it was the
most abundant protein among the extracelluar proteins. On the
basis of the tRES reaction profile, we hypothesized that that
such a tyrosinase degrades tRES via its ortho-hydroxylated
intermediate, i.e., piceatannol, and undergoes subsequent
melanine synthesis via its oxidized quinone form. Yang et al.
supported a similar idea by showing that the extracellular
tyrosinase (MelC2) of S. avermitilis converts phenol into
quinone via catechol.18
R
and clinical study on rats and humans showed that trans-
resveratrol (tRES) has excellent antioxidizing function and can
be further developed as anti-cancer, anti-inflammatory, and
blood-sugar-lowering supplements and drugs.4−7 Extraction
from natural resources and bacterial fermentation are two ways
to make resveratrol in the food and pharmaceutical industries.
For example, grape vines treated with cyclodextrin produce up
to 5 g L−1 tRES,8 and Lim et al. showed that 2.3 g L−1 of tRES
was obtained by combined biosynthesis of genes originating
from plants and bacteria in E. coli.9
Piceatannol is a C3′ hydroxylated product of resveratrol
found in plants (Figure 1). It is well-known as a tyrosine kinase
inhibitor and suppressor of cancer cell proliferation and
growth.10−13 At human liver microsomes, pro-drug tRES is
converted to piceatannol by cytochrome P450s such as
CYP1B1 and CYP1A2.14−16 To produce a high amount of
piceatannol, microbial P450s, e.g., BM3 mutants from Bacillus
megatrium are commonly used rather than recombinant CYP
1A2. Although the current production yield of BM3 mutants
from B. megatrium is quite low for industrial applications, few
researchers have been successful in screening microbial P450s
capable of performing its ortho-hydroxylation.17
We proposed to either clone MelC2 of S. avermitilis into E.
coli or evaluate tyrosinase deletion mutants of S. avermitilis.
However, we were unable to express recombinant tyrosinase
(MelC2) in E. coli, because functional expression of the
tyrosinase was strongly dependent on the helper protein
Various Streptomyces strains were evaluated to identify
microorganisms with such P450s to convert tRES into
piceatannol. By measuring the decrease in initial tRES
Received: May 8, 2012
Accepted: July 6, 2012
Published: July 6, 2012
© 2012 American Chemical Society
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Figure 1. Schematic representation of trans-resveratrol hydroxylation and the inhibition of piceatannol oxidation. Tyr, tyrosinase.
(MelC1) whose expression was not possible in E. coli.
Therefore, we evaluated the MelC2 deletion mutant.
RESULTS AND DISCUSSION
■
Screening of Streptomyces Strains for ortho-Hydrox-
In general, the tyrosinase reaction follows two steps in which
the monophenolic compound is converted into a dioxygenated
quinolic compound via the monooxygenated dihydroxy form.
The two copper ions centered in the tyrosinase are essential for
its activity,19−21 as the two copper ions hold one oxygen
molecule (oxy-tyrosinase), and subsequently a monophenolic
substrate is converted to a catechol-like molecule by bridging to
the copper ions of tyrosinase (met-tyrosinase). Finally, the
catechol-like molecule is converted to a quinolic compound
with synchronous formation of one water molecule (deoxy-
tyrosinase). However, it is difficult to detect the mono-
oxygenated dihydroxy compound intermediate in the reaction
mixture, because subsequent formation of the dioxygenated
quinolic compound is very rapid. Therefore, many researchers
have attempted to back-reduce the quinolic compound to the
dihydroxy compound to produce the dihydroxy compound
intermediate. At the moment of formation of the quinolic
compound, one oxygen atom of met-tyrosinase and a water
molecule withdraw electrons from the catechol-like molecule. A
reducing agent such as NADH,22 glutathione,23 or L-ascorbic
acid24 is required as an electron donor to prevent the catechol-
like molecule from subsequent oxidation. However, it is quite
costly to use such reducing reagents in an industrial-scale
production process. Another possible approach is protein
engineering of tyrosinase to reduce the dioxygenase reaction
rate. But, such attempts have not been successful, because the
ratio of the monooxygenase reaction to the dioxygenase
reaction of tyrosinase does not increase significantly.
Instead, here we propose that catechol-like molecules can be
used as effective inhibitors to adjust the tyrosinase dioxygenase
reaction such as the oxidation of piceatannol. This method of
using a tyrosinase inhibitor during the tyrosinase reaction
appears to be far superior to P450 monooxygenase for the same
reaction in terms of conversion yield and productivity, as the
kcat/Km values of tyrosinases are almost 10−100 times higher
than that of corresponding P450s.25 Additionally, unlike P450s,
no cofactors such as NAD(P)H are required for the enzyme
reaction, although their substrate specificities are somewhat
narrowly confined to aromatic catechol-like molecules.26 When
this tyrosinase reaction is successfully applied to ortho-
hydroxylation of catechol-like molecules, many useful dihy-
droxy aromatic compounds can be synthesized at high
concentrations, which is not easy to develop with mono-
oxygenases.
ylation of tRES to Piceatannol. Because Streptomyces contain
several P450 monooxygenases for (iso)flavonoid ortho-
hydroxylation,27,28 various strains were screened for ortho-
hydroxylation of tRES. After 100 μM tRES was added to the
reaction media containing 50 mL of phosphate buffer (pH 7.5),
the reaction was monitored every 3 h by detecting product
formation and substrate degradation. The piceatannol product
was identified by gas chromatography/MS (GC−MS) (see
Supplementary Data) and quantified by high performance
liquid chromatography (HPLC). Retention time and the MS
peak product pattern exactly matched those of authentic
piceatannol. Among the eight strains examined, four brown-
black melanin producing strains such as S. avermitilis, S. griseus,
S. pristinaespiralis, and S. venezuelae consumed almost all of the
tRES and produced ca. 5 μM piceatannol, whereas four
nonmelanin producing strains, S. caelestis, S. coelicolor, S.
peustius, and S. lividans, did not show any changes in the initial
tRES concentration (Figure 2), suggesting that melanin
synthesis in Streptomyces has a very close relationship with
the transformation of tRES to piceatannol.
Melanin and Piceatannol Production by a Secreted
Protein from S. avermitilis. S. avermitilis was selected to
identify the piceatannol-producing enzyme, because its genome
information was openly available, and the method of
constructing the recombinant strain has been established.
According to its genome database (http://avermitilis.ls.
kitasato-u.ac.jp/), 33 P450s and two tyrosinases in S. avermitilis
were putative candidates for piceatannol formation. One of the
two tyrosinases, MelC2, is a secreted protein that leads to
melanin formation outside the S. avermitilis cell,18 whereas
P450 hydroxylation occurs only intracellularly, as P450 is
normally supported by redox partner proteins such as
ferredoxin and ferredoxin reductase to transfer electrons inside
the cell.29,30 To identify which proteins are involved in both
melanin synthesis and piceatannol production, the activity of
intracellular and extracellular proteins were compared. tRES
biotransformation was executed using both the cell pellet
prepared by centrifugation and subsequent washes with 50 mM
phosphate buffer (pH 7.5), and the supernatant of the same
culture broth. As shown in Figure 3, the 50 mL cell supernatant
samples consumed all of the initially batched tRES within 30
min. In the case of the whole cell reaction, very slow but
marked reduction of tRES was observed. However, it was
unclear which was responsible for the reduction of tRES
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construction and expression levels of the helper proteins and
tyrosinases, reverse transcription-polymerase chain reaction
(RT-PCR) assays of all tyrosinases (MelC2 and MelD2),
tyrosinase helper proteins (MelC1 and MelD2), and four other
dioxygenases as a control were conducted with white ΔmelC2
and white ΔmelD2 mutants (Figure 4B and C). In the ΔmelC2
Figure 2. Comparison of consumption of 100 μM trans-resveratrol
transformed by whole cells of Streptomyces spp. (A) Melanin-
■
●
▲
producing Streptomyces spp.: ( ) S. avermitilis, ( ) S. griseus, ( ) S.
◆
pristinaespiralis, ( ) S. venezuelae. (B) Non-melanin-producing
Streptomyces spp.: ( ) S. caelestis, ( ) S. coelicolor, (Δ) S. peustius,
□
○
◇
(
) S. lividans.
Figure 3. Time profile of 100 μM trans-resveratrol converted by whole
cells, centrifuged, and subsequently washed twice with 50 mM
Figure 4. Characterization of Streptomyces avermitilis WT, ΔmelC2,
and white ΔmelD2. (A) SDS-PAGE of secreted proteins concentrated
10 times. Major band of wild-type (WT) was MelC (SAV1137). (B)
1.5% (w/v) agarose gel analysis of PCR product using cDNA of
tyrosinase, caddie proteins, and several dioxygenases. a: MelC1 helper
protein (SAV1136), b: MelC2 tyrosinase (SAV1137), c: MelD1 helper
protein (SAV5361), d: MelD2 tyrosinase (SAV5362), e: dioxygenase1
(SAV565), f: dioxygenase2 (SAV1615), g: dioxygenase3 (SAV2478),
h: dioxygenase4 (SAV5671), i: 5S RNA (control). (C) Relative
expression level of each mRNA compared to 5S RNA quantity.
■
●
phosphate buffer (pH 7.5) ( ) and the supernatant broth ( ) without
treating with reducing agents. It was difficult to quantify the
piceatannol concentration due to low peak area on the high
performance liquid chromatography (HPLC) chromatogram.
between the P450s and the secreted tyrosinase from the cell
pellet during the reaction. Tyrosinase deletion mutants were
constructed to clarify that the extracellular tyrosinase produced
not only piceatannol but also melanin.
Characterization of Tyrosinase Deletion Mutants of S.
avermitilis. To confirm which tyrosinase is involved in
piceatannol production, extracellular tyrosinase (MelC2) and
intracellular tyrosinase (MelD2) deletion mutants were
constructed with S. avermitilis (see Supplementary Data).
First, the color of each mutant was compared visually to
measure the degree of melanin synthesis. One half of the
ΔmelD2 transformants were white, and the other half were
brown, whereas all ΔmelC2 transformants were white. Brown
ΔmelD2 deletion mutants were excluded from further experi-
ments, because the levels of melanin synthesis of the brown
ΔmelD2 mutants were similar to wild-type (WT), indicating
that the brown color is naturally caused by the extracellular
tyrosinase activity of MelC2. To confirm the proper gene
mutant, only the melC2 gene was disrupted and melC mRNA
was not detected. However, in the case of ΔmelD2 mutant, not
only the melD2 gene was totally disrupted but also the melC
operon decreased significantly. PCR of the two genes was
carried out using the genomic DNA of the ΔmelD2 strain to
confirm whether its genomic DNA was damaged during the
deletion mutation. The band of the melC operon genes was
observed on an agarose gel (data not shown). Compared to
WT, the expression level of melC operon mRNA in the ΔmelD2
mutant was strongly suppressed approximately 104-fold,
whereas expression levels of the four other dioxygenases were
not affected by melC or melD gene deletion alone. As shown in
the SDS-PAGE gel of Figure 4A, MelC2 (30.4 kDa) secreted
from the cell was not observed in either white ΔmelC and
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ΔmelD mutants in contrast to WT. A possible explanation is
that the melC operon is somewhat inducible and that melC1/
melC2 expression is indirectly regulated by the presence of
MelD2. In the white ΔmelD2 mutant, not only monooxygenase
activity for tRES hydroxylation but also dioxygenase activity for
melanin synthesis were not shown. In both white melC2 and
melD2 deletion mutants, the piceatannol peak on the HPLC
chromatogram was not detected at all even after 24 h of
reaction with tRES. At the same time, the initial tRES
concentration did not decrease noticeably, suggesting that
MelC2 is the crucial enzyme for tRES hydroxylation. From the
result of tRES transformation by tyrosinase deletion mutants,
we conclude that extracellular tyrosinase (MelC2) is essential
for tRES hydroxylation, unless other tRES monooxygenases are
amplified. Although there is some possibility that such
intracellular P450s and other monooxygenases convert tRES,
considering their kcat/Km values and expression levels in the
host cells compared to those of MelC2, the monooxygenases
may not significantly contribute to tRES hydroxylation. For
example, the intracellular P450, CYP105D7 from S. avermitilis
expressed in E. coli could transform daidzein to 3′-ODI (o-
dihydroxyisoflavone) with a kcat/Km value of 0.0115 μM−1 s1−,25
which is similar to that of tyrosinase for tyrosine. However, in
the case of S. avermitilis the MelC2 expression level was much
higher than that of CYP105D7, because MelC2 is the major
protein in the cell supernatant and accumulated during the
fermentation and bioconversion (Figure 4A). Therefore, the
role of S. avermitilis monooxygenases in hydroxylation is minor
relative to tyrosinase due to its relatively low kcat/Km value and
low expression level. The MelC2 deletion mutant should be
used to perform a correct evaluation and avoid a possible high
background hydroxylation reaction exerted by the tyrosinase
MelC2 to screen a target monooxygenase toward specific
monophenolic substrate in Streptomyces strains.
Evaluation of Reducing Agents for Blocking Tyrosi-
nase Activity As a Catechol Oxidase. In general, tyrosinase
has monooxygenase and dioxygenase functions; hence, it
catalyzes monophenolic compounds to catechol-like inter-
mediates and to quinolic compounds sequentially. Dioxygenase
activity should be suppressed to produce piceatannol from
tRES, while maintaining its monooxygenase activity. Two
strategies were attempted to suppress the second consecutive
oxidation of catechol-like intermediates. First, quinolic
compounds were contacted with reducing reagents such as
NADH or L-ascorbic acid, which donate electrons to quinolic
compounds and drive the reduction reaction. The other
strategy is inhibiting the dioxygenase reaction by adding
stronger competitive inhibitors than the catechol-like inter-
mediates. Then, perhaps, the stronger competitive inhibitors
can be oxidized by tyrosinase instead of piceatannol during the
second oxidation. To examine these two strategies, 1 mM
concentration of various reducing reagents or inhibitors was
added with 100 μM tRES to 1 mL of S. avermitilis cells
resuspended in 50 mM phosphate buffer (pH 7.5) for 3 h
(Table 1). Non-phenolic inhibitors such as NADH and L-
ascorbic acid are well-known tyrosinase reductants. Our
inhibitor study showed that phenolic inhibitors such as catechol
resulted in a higher yield of piceatannol than that from
nonphenolic reductants. The major reason why nonphenolic
reductants are less efficient than phenolic inhibitors appears to
be significant consumption of the nonphenolic reductants by
other oxidants and oxidases in the cell mixture. As shown in
Table 1, the highest relative yield of piceatannol converted from
Table 1. Evaluation of the Ability To Accumulate
Piceatannol with Various Reductants
a
type
chemicals
relative production, %
without treatment
NADH
7.2
64.2
63.7
22.2
42.4
non-phenolic
L-ascorbic acid
glutathione
cysteine
hydroquinone
1-naphthol
p-coumaric acid
curcumin
97.1
3.7
32.5
7.4
phenolic
catechol
100.0
32.1
6.4
pyrogallol
ferulic acid
a
100 μM trans-resveratrol was reacted with 1 mM reductant and whole
cells of S. avermitilis in 50 mM Tris-HCl, pH 8.0 for 3 h.
100 μM tRES was obtained when hydroquinone and catechol
were added to the reaction mixture at the same time that tRES
was added. When 1 mM catechol was added to the reaction
with 100 μM tRES, the yield of piceatannol was 1.5 fold higher
than when adding nonphenolic reductants such as NADH and
L-ascorbic acid. Catechol-like molecules slow down only the
dioxygenase reaction, acting as a competitive inhibitor against
dioxygenase but not against monooxygenase. In addition, the
catechol-like molecules may stabilize piceatannol by donating
electrons to two copper ions bridged to the oxygen atoms on
the C3 and C4 positions of piceatannol. Thus, phenolic
compounds become a crucial additive for the high piceatannol
yield in this tyrosinase reaction.
Commercial mushroom tyrosinase was also used for
piceatannol production. Its tRES consumption rate was similar
to that of S. avermitilis tyrosinase. However, the ratio of
monooxygenase to dioxygenase activity by mushroom
tyrosinase seemed to be much lower than that of S. avermitilis.
Further study on the comparison of the reactivity ratio of each
tryrosinase should be carefully performed in depth.
High-Yield Production of Piceatannol by Adding
Catechol. The results shown above suggest that catechol is
the best competitive inhibitor and reducing agent for high yield
production of piceatannol from tRES. To determine the
optimum inhibitor concentration, 100 μM tRES was reacted
with a resuspended S. avermitilis cell pellet in 50 mM phosphate
buffer (pH 7.5) by varying the catechol concentrations from 10
to 104 μM (Figure 5). The higher the catechol concentration
used, the more piceatannol was yielded. However, an excessive
amount of catechol slightly reduced total tyrosinase activity.
Catechol at 1 mM concentration was optimal for the tyrosinase
reaction with 100 μM tRES. Under the optimal condition of 50
mL of 50 mM Tris-HCl buffer and 1 mM catechol, the
maximum yield of piceatannol reached a 78% conversion yield
after a 4 h reaction (Figure 6). By using this simple inhibitor in
the biotransformation of S. avermitilis secreting MelC2, a high
yield of regiospecific hydroxylation of tRES was achieved.
Similar to the suppression of tyrosinase activity resulting from
the high catechol concentration, an excessive amount of tRES
(>1 mM) inhibited the tyrosinase reaction due to substrate
inhibition (data not shown). Based on this strategy, adding
catechol to the tyrosinase reaction would be an efficient and
simple way to produce ortho-hydroxylated catechol derivatives
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CaCl2·2H2O, 0.003% (w/v) L-proline, 2 mL of trace element solution,
and 5 mL of 1 N NaOH.
Plasmid Construction and Transformation of S. avermitilis
MA4680. S. avermitilis genomic DNA was extracted with A G-spin
Genomic DNA Extraction kit and used as a PCR template. The
tyrosinase genes were amplified by PCR using a set of specific primers
(see Supplementary Data). The PCR product was cloned into the
pIBR25 expression vector. The shuttle plasmid was constructed with E.
coli JM110 (Novagen) for transformation in S. avermitilis. Disruption
of MelC and MelD was performed using a plasmid pSuperCos1 to
deliver the corresponding apramycin resistance gene cassettes (see
Supplementary Data). The cassette consisted of two PCR-derived
flanking regions in which suitable restriction sites were introduced. All
constructs were delivered into S. avermitilis by protoplast-mediated
transformation. The transformants were selected on R2YE/apramycin
medium for melC and melD deletion and further confirmed by RT-
PCR.
Figure 5. Relative production of accumulating piceatannol with
different concentrations of catechol. From left to right: 0, 101, 102, 103,
and 104 μM. respectively. [resveratrol]0 = 100 μM; reaction volume,
50 mL; reaction time, 3 h.
RNA Extraction and RT-PCR. RNA extraction was carried out
using an RNeasy Mini kit (Qiagen, Valencia, CA, USA) according to
the manufacturer’s instructions. The concentration and purity of the
extracted RNA were determined by measuring the A260 and the ratio of
A260 to A280, respectively. RT-PCR was performed using the primers
and the probes of the MelC, MelD, and 5S RNA sequences to quantify
the melC and melD mRNA transcripts (see Supplementary Data). The
detailed experimental procedure was described in our previous work.32
Streptomyces Whole Cell Reaction. Each strain was subcultured
by shaking test tubes containing 1 g of glass beads in 3 mL of R2YE
medium at 200 rpm and 30 °C for 2 days. One milliliter of cultured
cells was inoculated into 50 mL of fresh medium with 4 g of glass
beads in a 250 mL flask, and the flask was incubated at 200 rpm and 30
°C for 24 h. The cells were centrifuged at 4000 rpm for 10 min and
were washed twice with 50 mM phosphate buffer (50 mL, pH 7.5).
After another centrifugation, 5 g of wet weight cells were added to 50
mL of the same phosphate buffer containing 100 μM tRES. The
reaction mixture was kept on the shaking incubator at 200 rpm and 30
°C. A 1 mL aliquot was extracted with 1 mL of ethyl acetate. After
centrifugation at 13,000 rpm for 10 min, the upper phase was
evaporated in a cold vacuum chamber. The product was analyzed by
HPLC or GC−MS.
Piceatannol Accumulation by Catechol Treatment. To
determine the optimal concentration of catechol for reducing
piceatannol degradation, 0.1, 1, 10, and 100 mM catechol
concentrations were compared. Catechol was preincubated with the
whole cell mixture for 5 min before resveratrol was added.
Additionally, the culture broth supernatants separated from whole
cells were compared.
Figure 6. Comparison of piceatannol production between (A)
secreted proteins and (B) washed whole cells of Streptomyces
avermitilis treated with 1 mM catechol and 100 μM trans-resveratrol
○
●
in 50 mM Tris-HCl, pH 8.0: ( ) trans-resveratrol, ( ) piceatannol.
Analytical Method. Time-dependent consumption of resveratrol
and formation of piceatannol were monitored by HPLC analysis. The
analysis was performed on an Autochro-3000 (Young Lin) connected
with an UV−vis detector and a C18 column (4.6 mm × 150 mm). The
HPLC analysis conditions were as follows: mobile phase, 30% (v/v)
acetonitrile in H2O containing 0.1% (v/v) trifluoroacetic acid; flow
rate, 1 mL min−1; injection volume, 20 μL; detection wavelength, 325
nm. GC−MS was used to identify tRES and its metabolites. All
hydroxyl groups of the dried sample extracted with ethyl acetate were
converted to TMS (trimethylsilyl) derivatives by heating for 30 min at
60 °C with BSTFA. GC−MS was performed on a Thermo Scientific
Trace GC Ultra instrument connected to a Thermo Scientific
ITQ1100 MS spectrometer with a TR-5 ms SQC capillary column
(30 m × 0.25 mm i.d., 0.25 mM film thickness). The GC−MS analysis
was carried out as follows: injector temperature, 250 °C; temperature
gradient, 150 °C hold for 1 min, 10 °C/min up to 300 °C, 300 °C
hold for 5 min; MS operating mode, 70 eV electron ionization mode.
in the chemical industry. Further studies must be conducted to
improve conversion yield of beneficial catechol derivatives at
higher concentrations of target molecules. One such strategy is
to construct a recombinant strain to overexpress functional
tyrosinase.
METHODS
■
Chemicals. tRES, piceatannol, NADH, catechol, pyrogallol, 1-
naphthol, p-coumaric acid, ferulic acid, glutathiohe, cysteine, L-ascorbic
acid, hydroquinone, curcumin, and N,O-bis(trimethylsilyl)-
tyrifluoroacetamide (BSTFA) were purchased from Sigma-Aldrich.
All other chemicals and solvents were commercially available.
Bacterial Strains and Culture Conditions. S. avermitilis MA4680
and other Streptomyces strains were obtained from the Korea
Collection for Type Cultures. All strains used are listed in the
Supplementary Data. The cell culture protocol for all strains followed a
general procedure.31 The strains were cultivated on R2YE medium,
containing 10.3% (w/v) sucrose, 1% (w/v) D-glucose, 1% (w/v)
MgCl2·6H2O, 0.025% (w/v) K2SO4, 0.5% (w/v) yeast extract, 0.01%
(w/v) casamino acid, 0.57% (w/v) N-tris(hydroxymethyl) methyl-2-
aminoethanesulfonic acid, 0.005% (w/v) K2HPO4, 0.03% (w/v)
ASSOCIATED CONTENT
■
S
* Supporting Information
This material is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
■
Corresponding Author
*E-mail: byungkim@snu.ac.kr.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This research was supported by a National Research
Foundation of Korea (NRF) grant funded by the Korea
government (MEST) (No. 20110020418) and WCU (World
Class University) program through the National Research
Foundation of Korea (NRF) grant funded by the Ministry of
Education, Science and Technology (R322011000102130).
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