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: sohng@sunmoon.ac.kr;

Nguyen Huy Thuan

Email address: thuanbiochem@gmail.com

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