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Original research article World Journal of Microbiology and Biotechnology
Microbial Production of Astilbin, a Bioactive Rhamnosylated Flavanonol,
From Taxifolin
Nguyen Huy Thuan1*, Sailesh Malla2, Nguyen Thanh Trung1, Dipesh Dhakal3, Anaya Raj
Pokhrel3, Luan Luong Chu3 and Jae Kyung Sohng3,4*
1Center for Molecular Biology, Institute of Research and Development, Duy Tan University,
K7/25 Quang Trung, Danang, Vietnam.
2Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark,
Kemitorvet, 2800 Kgs. Lyngby, Denmark.
3Department of Life Science and Biochemical Engineering, 4Department of BT-Convergent
Pharmaceutical Engineering, SunMoon University, 70 Sunmoon-ro 221, Tangjeong-myeon,
Asan-si, Chungnam 31460, Republic of Korea.
(*): Corresponding authors:
Prof. Dr. Jae Kyung Sohng
Tel.: +82 41 530 2246; Fax: +82 41 544 2919.
Email address: [email protected];
Nguyen Huy Thuan
Email address: [email protected]
Abstract
Flavonoids are plant-based polyphenolic biomolecules with a wide range of biological activities.
Glycosylated flavonoids have drawn special attention in the industries as it improves solubility,
stability, and bioactivity. Herein, we report the production of astilbin (ATN) from taxifolin
(TFN) in genetically-engineered Escherichia coli BL21(DE3). The exogenously supplied TFN
was converted to ATN by 3-O-rhamnosylation utilizing the endogeneous TDP-L-rhamnose in
presence of UDP-glycosyltransferase (ArGT3, Gene Bank accession number: At1g30530) from
Arabidopsis thaliana. Upon improving the intracellular TDP-L-rhamnose pool by knocking out
the chromosomal glucose phosphate isomerase (pgi) and D-glucose-6-phosphate dehydrogenase
(zwf) deletion along with the overexpression of rhamnose biosynthetic pathway increases the
biotransformation product, ATN with total conversion of ~ 49.5 ± 1.67 % from 100 µM of
taxifolin. In addition, the cytotoxic effect of taxifolin-3-O-rhamnoside on PANC-1 and A-549
cancer cell lines was assessed for establishing ATN as potent antitumor compound.
Keywords: Biotransformation, cytoxicity, Escherichia coli, flavonoid, glycosylation.
Running title: Astilbin production in E. coli…
Introduction
Flavonoids are the most ubiquitous plant based polyphenolic secondary metabolites with a wide
range of biological activities (Falcone-Ferreyra et al. 2012; Miean et al. 2001). However, the
pharmaceutical applications of flavonoids are limited due to their low solubility, stability and
bioavailability because of their hydrophobic nature. Glycosylation is one of the promising post
tailoring functional modification of secondary metabolites in plants and other organisms, which
confers physical changes, including solubility and stability, as well as bioactivity (Kren et al.
1997; Ruffing et al. 2006). Likewise absorption, distribution, metabolism, and excretion of drugs
as well as detoxification of exogenous compounds can be greatly enhanced by glycosylation
(Thuan et al. 2013c; Kren et al. 2003; Kren et al. 2001). Hence, among various flavonoid
derivatives, glycosylated forms have drawn special attention in the industries as it improves
solubility, stability, and functionality (Ghimire et al. 2015; Griffith et al. 2005; Ahmed et al.
2006; Gantt et al. 2011). Astilbin (ATN), also known as taxifolin (TFN)-3-O-rhamnoside (Fig. 1)
is a rhamnosylated flavanonol which has been used in traditional Chinese medicine (Zhang et al.
2010; Huang et al. 2011). ATN is mainly isolated from a commonly used herbal medicinal plant,
Smilax glabra Roxb (Zhang et al. 2010). In addition, ATN is also found in Astilbe thunbergii,
Astilbe odontophylla (Saxifragaceae), Dimorphandra mollis, Harungana madagascariensis,
Hymenaea martiana, Hypericum perforatum, etc.
Currently, plant extract is the only source of ATN, however, the industrial scale production by
plant extracts for drug development is still a major challenge (Ren et al. 2012; Oleszek et al.
2002; Prawat et al. 2012), though there have been significant studies for optimization of
extraction condition (Lu et al., 2015). The traditional chemical synthesis of natural products
relies mainly on energy-intensive conversions of petroleum-derived carbon feedstocks (Tang et
al. 2004). In addition, the activated sugars must be used in stoichiometric ratio during chemical
synthesis making the prohibitively expensive process. On the other hand, biobased industrial
processes (i.e. microbial fermentation) allow the conversion of renewable-carbon feedstocks into
varieties of chemical compounds at comparatively low temperatures and pressures (Park et al.
2010; Cho et al. 2014). Before microbial host were used to synthesize taxifolin glycosides, these
compounds were extracted from high plant, for instance, neoastilbin from leaves of Engelhardtia
chrysolepis (Kasai et al. 1998) or synthesize using plant tissue culture, for example, cultured
plant cells of Eucalyptus perriniana glucosylated taxifolin to its 3'- and 7-O-β-D-glucosides and
3',7-O-β-D-diglucoside. On the other hand, taxifolin was also converted into 3'- and 7-O-β-D-
glucosides by cultured cells of Nicotiana tabacum and Catharanthus roseus (Shimoda et al.
2013).
Herein, we have reported an efficient and sustainable process to generate ATN from taxifolin, a
flavanonol, in metabolically engineered E. coli BL21(DE3) strain. The bioconversion product
was confirmed by high performance liquid chromatography photodiode array (HPLC-PDA) and
liquid chromatography - electrospray ionization mass spectrometry (LC–ESI/MS. The detailed
structural elucidation was performed by 1D NMR (1H and 13C nuclear magnetic resonance
(NMR) studies) and 2D (2-Dimension) NMR studies. The production was scaled up in 3 L
fermenter. Furthermore, the cytotoxic activity of the purified ATN was tested for a human lung
epithelial (A549) and epithelioid carcinoma (PANC1) cell lines.
Materials and Methods
Culture media and chemicals
LB (Luria-Bertani) and TB (terrific broth) were used for seed culture and substrate fermentation.
Antibiotics including ampicillin, kanamycin and chloramphenicol (Biobasics, Canada) were used in the
final concentration of 100, 50 and 30 µg/mL, respectively. Ethyl acetate, methanol, toluene,
dimethylsulfoxide, etc were purchased from Merck (Germany) or Sigma (USA). Nanodrop 2000 UV-Vis
Spectrophotometer (Thermo, USA), HPLC-PDA (Agilent, USA) and NMR (Bruker, USA) were used for
chromatographic analysis.
Bacterial strain, DNA manipulation and plasmids
A laboratory host strain E. coli BL21 (DE3) was used. Construction of deletion mutant was
earlier described in earlier studies (Malla et al. 2013, Pandey et al. 2013; Thuan et al., 2013a).
Construction of plasmids pCD-TGSDH, pAC-EPKR and pET28-ArGT3 (Gene bank accession
number: At1g30530) was described previously (Simkhada et al. 2010). All DNA manipulations
were carried out by following standard protocols (Sambrook et al. 2001).
Biotransformation process and product isolation
Pre-cultures of the engineered E. coli strain were carried out with initial volume of 3 ml broth
culture in 15 ml falcon tube at 37 ºC, until the optical density (OD600nm) ~ 1 measured by
spectrophotometer. Subsequently, 0.5 ml of the culture broth was inoculated in flask 250 ml
containing 50 ml medium (i.e., 1:100 dilutions). The culture flasks were incubated at 37 oC, 220
rpm until it reached OD600 ~ 0.6. Subsequently, 1 mM of Isopropyl β-D-1-thiogalactopyranoside
(IPTG) was added to induce protein expression and the flasks were incubated at 32 oC and 220
rpm for the next 3 h. Then, 100 µM of taxifolin dissolved in dimethyl sulfoxide (DMSO) was
supplied to the cultures and continued incubation. Samples (3 ml of culture broths) were
withdrawn at 24, 36, 48 and 60 h for product analysis. The samples were extracted with double
volume of ethyl acetate. The organic layer was collected and the solvent was evaporated to
dryness. The resultant concentrate was dissolved in 1 mL methanol for high performance liquid
chromatography (HPLC-PDA) and liquid chromatography–electrospray ionization mass
spectrometry (LC–ESI/MS) analysis.
Analysis and quantification
HPLC-DAD analysis was performed by injecting 15 µl of the samples on an Agilent 1260 HPLC
system equipped with a photodiode array detector (DAD), degasser, and autosampler. An
Agilent Zorbax SB C18 column (250 mm × 4.6 mm i.d., 5 μm; Agilent, Santa Clara, CA, USA)
was used. The mobile phase of 0.1% trifluoro acetic acid (TFA) aqueous solution (solvent A)
and acetonitrile (solvent B) were used with 1 ml/min flow rate. The concentration of acetonitrile
during the binary gradient condition was as: 0-15 min, 0-50%; 15-20 min, 50%; 20-25 min, 50-
90%. Peak detection was carried out at UV absorbance at 285nm whereas. Under these conditions,
the retention time for taxifolin was 9.2 min and for astilbin it was 8.5 min.
Astilbin was purified using a MPLC instrument equipped the silicagel RP-packed column (YMC
gel ODS-A, AA12SA5, Japan). The purified product was lyophilized. A series of concentrations
ranging from 10 to 100 mg/L of product were prepared to construct calibration curve (Fig. S1).
Molecular mass of the compounds were determined in LC-ESI-MS using Phenomenex Synergi
Polar-RP column (150 × 4.6 mm, 4 µm), negative-ion mode.
Structural elucidation of astilbin
The lyophilized sample of the purified HPLC peak (corresponding to 8.5 min) was dissolved in
DMSO-d6 (Sigma-Aldrich) then 1H and 13C nuclear magnetic resonance (NMR) spectra were
recorded by using NMR Bruker advanced instrument (500 MHz). NMR spectra were analyzed
by using MestReNova 8 program (Mestrelab Research S.L., Spain). The structure of ATN was
determined based on the interpretation of the NMR data (Silva et al. 1997; Batista et al. 2002).
LC-ESI-MS and NMR analysis were carried out in the Center for Applied Spectroscopy,
Institute of Chemistry, Vietnam Academy of Science and Technology (VAST).
Scale-up of Biotransformation System in Fermenter
Fed batch fermentation was carried out in BioTron equipment (BioTron Ltd., Incheon, Republic
of Korea) fitted with 5 L vessels. The temperature, pH and rotor speed were constantly
maintained at 32 oC, 7.5 and 400-500 rpm, respectively. 3 L of TB media supplemented with 3%
D-glucose (w/v) and 1% mannitol (w/v) was used as starting fermentation media. Dissolved
oxygen (DO) was maintained above 70% throughout the experiment. When OD600 reached 5, the
culture was induced by L-lactose with a final concentration of 0.15 M. After 3-4 h of
fermentation, 5.0 ml of D-glucose (100 g/L concentration) was fed every hour. The fermentation
broth was harvested after 60 h. Three milliliters of the harvested broth was subjected for HPLC
analysis as described above and the remaining culture broths were extracted and purified (as
described above) for NMR analysis.
Cytotoxicity assay
Human lung carcinoma cell line (A549) and human pancreatic carcinoma cell line (PANC-1)
were cultured in RPMI 1460 or DMEM media (Gibco, USA) containing 10% Fetal Bovine
Serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin, respectively. All cells were
maintained at 37 oC in a humidified 5% CO2 incubator. For growth assay, 2x103 cells/well onto
96-well plates (SPL Lifesciences, Gyeonggi, Korea) were treated with compound under study at
various concentrations for 72 h. Cell growth was measured using a 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT, Duchefa Biochemie, Netherland) colorimetric assay.
Optical density was measured at 570nm. Etoposide was used as positive control for the assay. Cell
survival percentage (% CS) was calculated by the followed formula: % CS= {(At-Ab)/ (Ac-Ab)}
x100 where, At= Absorbance value of test compound; Ab= Absorbance value of blank and
Ac=Absorbance value of control; Cell inhibition percentage (% CI) = (100-CS) %. This
experiment was done in the Department of Bioactive Products, Institute of Marine Biochemistry
(IMBC), Vietnam Academy of Science and Technology (VAST).
Statistical analysis
The Student’s t test was performed on the biological replicates to determine the statistical
significance of the difference between control and experiment samples at each time point.
Differences with P value < 0.05 were considered statistically significant.
Results
Engineering TDP-L-rhamnose pathway and whole cell biotransformation to ATN
For engineering the TDP-L-rhamnose pathway pathway, the chromosomal genes glucose-6-
phosphate isomerase (pgi) and glucose-6-phosphate dehydrogenase (zwf) were knocked out to
accumulate the pool of glucose-6-phosphate (G6P). Further, genes encoding for TDP-glucose
synthase (TGS) from Thermus caldophilus GK24, TDP-glucose 4,6-dehydratase (DH) from
Salmonella thyphimurium LT2, and TDP-4-keto-6-deoxyglucose 3,5-epimerase (EPi) and TDP-
glucose 4-ketoreductase (KR) from Streptomyces antibioticus Tü99 were overexpressed under
the control of strong T7 promoters, respectively. The engineered E.coli cell was transformed
with a recombinant plasmid pET28-UGT78D1, i.e., putative UDP-glucose:flavonoid-3-O-
glucosyltransferase from Arabidopsis thaliana (Gene Bank accession number AF360160) cloned
under T7 promoter in pET28a(+) vector (Simkhada et al. 2010). This host strain was named as E.
coli M3G3 (Thuan et al. 2013a).
The E. coli M3G3 was subjected for bioconversion process at 32 oC, 220 rpm and initial pH of
7.5. The biotransformation system was induced with 1 mM of IPTG and 100 µM of taxifolin
(aglycone moiety) was supplied into the cell cultures. Taxifolin was supplemented into
recombinant E. coli culture broth for investigating effect of this flavonoid concentration on the
bioconversion of substrate. Various concentrations of taxifolin including 0, 40, 60,100, 150 and
200 µM were fed to the recombinant E. coli broth culture. The percentage (%) of bioconversion
and effect on cell growth was measured and result is illustrated in the Fig S2. It was observed
that 100 µM was suitable concentration for bioconversion without significant effect on cell
growth.
The production of ATN (i.e., taxifolin-3-O-rhamnoside) and optical density of the cultures
(OD600nm) were analyzed at regular interval of time profile. With few hours of lag phase (data not
shown), OD value increased linearly up to 48 h and then there was remarkable decreased in cell
growth probably due to the lowering in pH by acetate formation. Preliminarily, the cell extracts
were HPLC analyzed which showed the production of ATN corresponding to the peak at
retention time of 8.5 min whereas the aglycone moiety taxifolin gives peak at 9.2 min (Fig. 2A).
Subsequently, the peak at 8.5 min was analyzed in LC-ESI-MS in negative mode which showed
that [M-H]-, m/z = 448.9, corresponding to O-rhamnosylated taxifolin (Fig.2B). The time
dependent bioconversion analysis showed that ATN was observed after 12 h of the substrate
supplementation into the induced system. Then the product concentration increases with time
which reached maximum 33.5 ± 1.3% conversion (i.e. 33.5 ± 1.3 µM or i.e. 15.1 ± 0.25 mg/L of
ATN production)) at 48 h (Fig. 3A). Hence, these results showed that the E. coli strain harboring
the biosynthetic genes for overproduction of TDP-L-rhamnose and GT can efficiently convert
taxifolin to its rhamnoside derivative in-vivo.
Structural confirmation of ATN
For structural elucidation of O-rhamnosylated taxifolin, in 500 ml of baffled flask bioconversion
reaction was carried out in 100 ml of media supplemented with 1% (w/v) mannitol and 3% (w/v)
glucose to increase the yield of substrate bioconversion (Thuan et al. 2013b; Koirala et al.
2014a). After 60 h, the product was isolated and purified using MPLC, dried and subjected for
structural elucidation. The obtained structure of ATN was then analyzed by 1D-NMR (1H, 13C
NMR), 2D-NMR (COSY, HMBC and HSQC) and compared to published data (Table 1, Fig S3
and Fig S4).
The rhamnosyl residue in the purified product corresponding to the HPLC peak at 8.5 min and
mass [M-H]-, m/z = 448.9. This spectrum showed an H-bonded hydroxyl group assigned to the
signal at 11.9, two doublets for H-2 and H-3, respectively, at 5.24 and 4.65 (J=10.0 Hz) along
with two signals for H-6 and H-8 of ring A 5.90 (d, J=2.0 Hz), 5.88 (d, J=2.0 Hz). The hydrogen
signals of ring B are [6.88, (br, s, H-2’) 6.73 (br, s, H-5’,6’)]. The 13C NMR data contained 21
carbon signals, 15 of which were typical for a flavononol (taxifolin) skeleton and six were
assigned to a rhamnose moiety. The rhamnose moiety was confirmed to be attached to the C-3
position of the aglycone by interaction between H-3 with C-1” and H-1” with C-3 as shown in
HMBC data (Silva et al. 1997; Batista et al. 2010; Lou et al. 1999) (Fig S4).
Scale up by fed batch fermentation
To test the reproducibility of the bioconversion process into the large-scale, fed batch
fermentation of the E. coli M3G3 under aerobic condition in 3 L volume at 32 °C was carried out
supplying taxifolin (100 µM). The pH of the fermentation was maintained in between 7.0-7.5
throughout the process to reduce growth inhibition caused by acetate formation. At every 4 h of
regular interval, culture broths were withdrawn and the samples were analyzed by HPLC-DAD.
The HPLC analysis of the fermentation samples showed that ca. 1.47 fold of improvement in
bioconversion process with 49.5 ± 1.67 % conversion (49.5 ± 1.67 µM i.e., 22.3 ± 0.5 mg/L of
ATN production) at 48h (Fig. 3B). Hence, the bioconversion titer was noticeably increased by
supplying 1% (w/v) mannitol and 3% (w/v) glucose into pH controlled TB media.
Cell cytotoxicity testing
In addition to the previously reported bioactivities of ATN, we have also studied the cytotoxic
effects of taxifolin, ATN (taking 30 and 100 µM concentrations) using a human lung epithelial
cell line (A549) and an epithelioid carcinoma cell lined (PANC1). Etoposide was taken as
positive control the bioactivity test. We observed the inhibition of 50% of A549 cell in presence
of 30 µM ATN which increases to 65% at 100 µM of its concentration (Table 2). The IC50 of
ATN for A549 was 30,5 ± 1,16 µM (Table 3).
Discussions
The whole cell biotransformation process wherein substrate molecule such as supplemented
flavonoids leading to spontaneous modification by the cells' endogeneous cofactor(s) and
enzymes is one of the easiest ways to attain structurally diverse products (Cao et al., 2014).
Escherichia coli (E.coli), by virtue of its ability to grow in high cell density within a short
production cycle and high substrate conversion even by consuming low energy, have been used
commonly used as platform organism for production and modifications of different natural
products (Gupta and Shukla, 2015). E. coli have great ability to uptake externally supplied
substrates/compounds and metabolize into its derivatives. Such bioconversion properties have
been extensively used for glycosylation, hydroxylation, methylation etc. of small molecules
(Williams et al. 2011; Leonard et al. 2005; Malla et al. 2012; Koirala et al. 2014b).
Recently, numerous studies have proven that the fermentation of metabolically engineered E.
coli strains expressing glycosyltransferase (GT) have produced wide ranges of glycosylated
flavonoids achieving large quantities for industrial applications. For example, Singh et al. (2013)
synthesized flavone-3-O-glucoside using GT (UGT73A16) from Withania somnifera (Singh et
al. 2013). Various glucosides of flavones- (daidzein), isoflavones (flavopiridol), stilbenes
(resveratrol) have been generated by OleD GT from Streptomyces (Zhou et al. 2013). In another
study, puerarin glucosides have been synthesized by GT from Leuconostoc dextransucrase (Ko
et al. 2012). Similarly, there are reports of several flavonoid glycosides such as kaempferol-3-O-
glucoside (Malla et al. 2013), quercetin 3-O-xyloside (Pandey et al. 2013), quercetin-3-O-
rhamnoside (Simkhada et al. 2010), myricetin-3-O-rhamnoside (Thuan et al. 2013a), flavone-7-
O-glucosides (Thuan et al. 2013b) or apigenin-O-glucosides (Gurung et al. 2013), quercetin 3-O-
galactoside (De Bruyn et al. 2015) in genetically engineered E.coli strains. Furthermore, a
chimeric gene was constructed by combining two Arabidopsis GTs (AtUGT78D2 and
AtUGT78D3) to enhance catalytic efficiency and extended sugar donor selectivity using
quercetin as acceptor (Kim et al. 2013).
Taxifolin is flavanonol molecule possessing remarkable biological activities (Topal et al. 2016;
Zhang et al., 2010; An et al., 2008). To further improve its pharmacological properties as well as
stability by using regiospecific glycosyltransferases (GTs), at first we engineered the metabolic
pathways of the host strain, E. coli BL21 (DE3), to direct the carbon flux for increasing TDP-L-
rhamnose pool (Thuan et al. 2013a). The glucose in the culture media is converted into glucose-
6-phosphate (G6P) which is a common precursor for the formation of fructose-6-phosphate
(F6P) and 6-phosphogluconate (6PG). Two chromosomal genes; glucose-6-phosphate isomerase
(pgi) and glucose-6-phosphate dehydrogenase (zwf) were knocked out to improve the flux flow
toward G1P from G6P (Malla et al. 2013). Thus accumulated G6P was channelled to TDP-L-
rhamnose by overexpressing TDP-glucose synthase (TGS), TDP-glucose 4,6-dehydratase (DH),
and TDP-4-keto-6-deoxyglucose 3,5-epimerase (EPi) and TDP-glucose 4-ketoreductase (KR).
This engineered double mutant strain with overexpressed rhamnose pathway is able to produce
ca. 8.8 fold higher (112.3 μM) of TDP-L-rhamnose as compared to that of the E. coli BL21
(DE3) (12.8 μM), respectively (Thuan et al. 2013a). The produced activated sugar (i.e. TDP-L-
rhamnose) can be attached into the target aglycone moiety in presence of favorable GT. The
specific rhamnosylation at 3-OH of TXN is able to generate resonable titer of ATN.
Furthermore, supplementation of carbon source as mannitol and glycerol was effective for
increasing the yield from fermentation process. This increased yield of production was in
agreement with our previous publications (Pandey et al. 2013; Thuan et al. 2013b). However,
according to Zhang et al. (2013), astilbin was less stable in culture broth compare to water which
may be related to the presence of metal ions, hence it can be speculated that real bioconversion
may be higher than the overall yield in the fermentation..
ATN is one of the most extensively studied flavonoids for its bioactivities. Modern
pharmacological studies showed that ATN has broad pharmacological properties such as
antibacterial (Moulari et al 2006), antioxidant, scavenging-free radicals (Petacci et al. 2010),
anti-inflammatory, assisting burn wound healing, anti-arthritic (Cai et al. 2003), anti-hepatic
(Wang et al. 2004), anti-renal injury (Chen et al. 2011), antidiabetic (Li et al. 2009) and enhance
immune function, etc. ATN also has neuroprotective effects suggesting its applicability for
treating Alzheimer's disease (Wang et al. 2016). It is also used as an insecticide (Batista Pereira
et al. 2002). In our study as well, ATN showed cytotoxic effects on human lung epithelial cell
line (A549) and an epithelioid carcinoma cell lined (PANC1). ATN has better activity on A549
as compared to that of taxifolin, which could inhibit about 30% of the cell growth at 30 µM
concentrations. This result suggests the applicability of ATN for treating lungs cancer. However,
in case of the PANC1 cell line, taxifolin showed better inhibition than that of the ATN. The
rhamnosylation plays an important role in the structural stability, solubility improvement,
intracellular transport, and bioavailability regulation of the natural products (Mo et al. 2016),
which may be leading factor for increased bioactivities in comparison to their corresponding
aglycons.
Due to immense biological importance of rhamnosylated derivatives, attempts have been
directed toward generating the rhamnosylated flavonoid by host engineering (Kim et al. 2012;
Parajuli et al. 2015). Recently, Mo et al. (2016) performed extensive study about aglycon
promiscuity and catalysis characteristics of AtUGT78D1 and created divergent rhamnose
conjugated natural products belonging to different structural types by rigorous in-vitro reactions
(Mo et al. 2016). However, despites its various health benefits, studies on the ATN for its
industrial production via microbial fermentation have not been reported, till date. This study is
first approach for the sustainable production of ATN using E. coli fermentation and assessment
of biological impact rendered by rhamnosylation in comparison to aglycon. In context to
evidence of greater substrate flexibility of the rhamnosyltransferase, this study depicts that
rational approach of generating different rhamnosylated derivatives of natural products and
assessing their comparative biological activities such as cytotoxicity can lead to discovery of
new drug leads.
Conclusions:
We have successfully engineered the E. coli strain for sustainable ATN production and showed
that the bioconversion process for its production could easily be scaled up by illustrating a model
study of 3 L fermentation. Besides the most of the reported activities for ATN, we evaluated its
activity against different cancer cells and obtained higher potential against lung cancer cells.
Further studies including the downstream process optimizations need to be carried out for the
industrial scale production. Hence this study provides evidence that an engineered microbial
platform can be utilized for production of the bioactive molecule such as ATN. This microbial
platform can be further fine-tuned for the production of novel derivatives of ATN or other
natural products as well. The optimization of bioprocessing parameters and rational engineering
of host using various synthetic biological tools and metabolic engineering can be rational
approach for obtaining such novel derivatives of different natural products (Dhakal and Sohng,
2015; Dhakal et al. 2016). Furthermore, the efficacy of ATN as an antitumor molecule against
lung cancer provides substantial background for targeted structure-activity relationship (SAR)
studies of such molecules.
Conflict of interest
The authors declare that they have no competing interests
Acknowledgements
This research was supported by the International Foundation for Science, Stockholm, Sweden,
through a grant to Nguyen Huy Thuan, grant number: F/5547-1, the National Foundation for
Science and Technology Development (NAFOSTED) of Vietnam [106-NN.02-2014.25] and 1
and by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ01111901),
Korea through a grant to Jae Kyung Sohng. We sincerely thank Dr. Nguyen Tien Dat
(Department of Bioactive Products) and Dr. Nguyen Xuan Cuong (Lab of Marine Medicinal
Materials), Institute of Marine Biochemistry (IMBC), Vietnam Academy of Science and
Technology (VAST), 18 Hoang Quoc Viet, Hanoi, Viet Nam) for assisting with the bioactivity
test and analysis of NMR data, respectively.
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Table Legends
Table 1. (A) 1H NMR of Astilbin (ATN) (800 MHz. DMSO-d6) and (B) 13C NMR (125 MHz,
DMSO-d6) of ATN.
Table 2. Cytotoxic assay of studied flavonoid on A549 and PANC-1. Data were expressed as the
mean±standard deviation (SD) of triplicates. Statistical analyses were performed with
GraphPad Prism Software.
Table 3. IC50 of tested substrates. Data were expressed as the mean±standard deviation (SD) of
triplicates. Statistical analyses were performed with GraphPad Prism Software.
Figure Legends
Figure 1. Schematic representation of E. coli cell factory (biotransformation system) design for
the production of astilbin (ATN) from taxifolin (TFN) and D-glucose. The chromosomal pgi and
zwf genes are were knocked-out, TGS and DH were overexpressed by cloning into pCDF-Duet
vector, EPi and KR were overexpressed by cloning into pACYC-Duet vector and the ArGT3 was
expressed by cloning into pET-28a(+) vector.
Figure 2. HPLC Chromatogram and mass analysis of the bioconversion product from E. coli
M3G3. A) HPLC-DAD analysis. i) authentic sample of taxifolin (TFN) peak shown by at 9.2
min retention time , ii) authentic sample of astilbin (ATN) peak shown by at 8.5 min retention
time, iii) Bioconversion product of 100 µM of TFN into ATN from E. coli M3G3 at 48h
incubation, and iv) E. coli BL21(DE3) control. B) LC-ESI-MS of ATN (rhamnosylated taxifolin)
in negative mode.
Figure 3. Production profile of ATN from taxifolin (100 µM supplementation) by whole cell
biotranformation of E. coli M3G3 (A) in batch cultures and (B) in fed batch fermentation. The
experiments were performed in biological triplicates. The error bars indicate the standard
deviations of the means of triplicates.
Table 1.
C Astilbin NMR spectrum (Lou et al. 1999) ATN spectrum data in this studyaδC
bδC δCc,d δH
c,e mult. (J in Hz)2 81.48 5.23 (d, J 9.8 Hz) 81.49 5.24 d (10.0)3 75.62 4.63 (d, J 9.8 Hz) 75.61 4.65 d (10.0)4 194.30 194.49 -5 163.41 163.39 -6 96.06 5.87 (d, J 2.0 Hz) 95.98 5.90 d (2.0)7 167.23 166.91 -8 95.11 5.89 (d,J2.0 Hz) 95.00 5.88 d (2.0)9 162.14 162.15 -
10 100.68 101.00 -1 126.97 126.91 -2 114.72 6.88 (s) 114.73 6.88 br s3 145.10 145.13 -4 145.90 145.87 -5 115.31 6.74 (s) 115.31 6.73 br s6 118.88 6.74 (s) 118.86 6.73 br s
5-OH 11.8 - 11.80 sRha1 100.02 4.04 (s) 100.03 4.04 s2 70.39 3.09±3.91 (m) 70.09 3.333 70.10 3.09±3.91 (m) 70.40 3.404 71.62 3.09±3.91 (m) 71.62 3.13 5 68.95 3.09±3.91 (m) 68.95 3.886 17.73 1.04 (d, J 5.9 Hz) 17.70 1.05 d (6.0)
aH and bC data of astilbin as followed by Lou et al. 1999, cDMSO-d6 as used as solvent, d125 MHz, e500MHz.
Table 2
Samples Concentration (µM) Cell viability (%)A-549 PANC1
Taxifolin 30 71.50±1.27 73.97±1.23100 41.53±0.93 50.38±0.97
Astilbin30 51.24±1.56 64.63±0.58
100 35.38±0.78 62.96±0.32Etoposide* 10 44.79±1.67 23.24±1.98
40 27.85±1.36 9.76±1.59Negative control 100 ± 1.2 100 ±0.79
Table 3
Samples IC50 (µM)A-549 PANC1
Astilbin 30.5±1.16 -Etoposide* 2.68±0.89 0.084±0.11Negative control 100 ± 1.12 100 ±0.77