Supplementary Information

Towards the production of flavone-7-O-β-D-glucopyranosides using

Arabidopsis glycosyltransferase in Escherichia coli

Nguyen Huy Thuan, Je Won Park and Jae Kyung Sohng٭

Department of Pharmaceutical Engineering, Institute of Biomolecule Reconstruction, Sun Moon University, #100,

Kalsan-ri, Tangjeong-myeon, Asan-si, Chungnam 336-708, Republic of Korea

D1. Construction of recombinant strains

E. coli BL21(DE3) (Novagen, Darmstadt, Hesse, Germany) containing UDP-glucose

biosynthetic pathway was utilized for deletion of zwf, pgi and ushA, as well as for combination

with over-expression of galU to generate various types of strains. The reconstruction process

resulted in E. coli BL21(DE3) (control), E. coli BL21(DE3)/∆pgi/galU, E. coli

BL21(DE3)/∆pgi/∆zwf/galU and E. coli BL21(DE3)/∆pgi/∆zwf//∆ushA/galU, which were used

as hosts [18]. All these strains were then transformed with recombinant pFL containing

Arabidopsis GT, to construct whole-cell biocatalysts which were named as strain MAF, MA1F,

MA2F and MA3F (Table 1).

D2. Cloning and expression of recombinant glycosyltransferase in E. coli host

cDNA of A. thaliana glycosyltransferase (RAFL15-41-P03) was purchased from RIKEN

Bioresource Center (NIG, Ibaraki, Japan) [4, 5]. All restriction enzymes and T4 DNA ligase

were obtained from Promega (Fitchburg, WI, USA) and Takara (Otsu, Shiga, Japan).

Oligonucleotide primers (Genotech Ltd., Daejon, Chungnam, South Korea) include 5’-TAC

GGATCC GAAATGGAGCCAAAGTTT-3’ (forward) and 5’-ACA AAGCTT

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TAATCAAATCAAATTCTC-3’ (reverse) containing recognized sequence of BamHI and

HindIII as restriction sites, respectively. GT was amplified by PCR, and was inserted into

pGEM-T-easy subcloning vector (Promega, Fitchburg, WI, USA) for sequencing. This DNA

fragment was then ligated into the pET32a(+) expression vector to obtain pFL plasmid, and was

finally transformed into E. coli to construct the recombinant host (Table 1).

D3. Media optimization

TB media containing 0.4, 1.0, 1.5 and 2% (w/v) glycerol were used as various media recipes. In

a similar manner, glucose-supplemented TB media with 0, 1, 3 and 5% (w/v) were also utilized

to culture the recombinant E.coli. In contrast, neither glycerol–supplemented nor glucose–added

TB media were used as controls. OD600 was then monitored at intervals of 12h to evaluate the

growth of E. coli.

D4. Extraction, purification and detection methods

The crude extract was obtained by adding of culture broth with 2 volumes of ethyl acetate (v/v =

2:1), stirred and dried by frozen rotary evaporator. The fat content was eliminated by dissolution

in the mixture of hexane and methanol (v/v = 1:5). The presence of the flavone glucosides was

then detected by thin layer chromatography (TLC) using the solvent system: ethyl acetate, water,

methanol and toluene (v/v/v/v = 8:1:1:0.2) in aluminum oxide plate were used as the initial

materials. Substrates and their glucosides were then detected by UV visualization at 280 nm.

They can also be detected as yellow spots by spraying with 10% sulfuric acid, and heating at

110°C for 5 s.

The products were purified in two steps. Firstly, each crude extract was partially fractionized

through a column (2.5 x 50 cm, Pyrex, Incheon, Sutogwon, Korea) filled with normal-phase

silica (Silica gel 60 GF254, Merck, Darmstadt, Hesse, Germany) to obtain target fragment, which

was then checked by TLC. Secondly, it was applied to the prep–HPLC system using an ODS

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column (2.5 x 25 cm, YMC-Pack ODS-AQ, Tokyo, Japan), and a UV detector at 280 nm. The

harvested fragment was then filtered twice using Whatman® paper (ɸ 0.2nm, Maidstone, Kent,

ME16 LS, England) and was finally freeze-dried.

A HPLC (Shimadzu, Kyoto, Japan) detection method was performed by binary program with

solvent systems, including acetonitrile (100%) as solvent B, and double distilled water (0.1%

formic acid) as solvent A. The program was described as follows: solvent B: 5% in 0–7 min, 5–

30% in 7–15 min, 30–90% in 15–25 min and 90–100% in 25–30 min. The solvent flow rate was

1 mL.min-1 and UV wavelength = 280 nm. The samples were then subjected to

LC-QTOF-ESI/MS (Water, Milford, MA, USA) to determine the mass of apigenin-7-O-

glucoside and baicalein-7-O-glucoside, respectively.

D5. NMR analysis of apigenin-7-O-β-glucoside (APG) and baicalein-7-O-β-glucoside (BCG).

The 1H-NMR spectra displayed a flavonoid pattern and showed signals at δ = 6.83 - 7.02 (1H, s)

6.83-7.07 (1H, s) and 6.46 or 8.6 ppm (1H, s) typical of protons at C-3, C-8 and C-6 of a flavone

core structure. The signal at δ = 12.57 or 12.98 ppm was assigned to the C-5 hydroxyl. 1H-NMR

resonances at δ 3.20 to 3.70 ppm and signals in the 13C-NMR spectrum just below δ = 70 ppm

indicated the presence of a glucose moiety [1]. The signal at δ = 5.15 ppm was assigned to the

anomeric proton (H-1”) with a coupling constant (J = 8.0 Hz) indicating a β-configuration [2]. In

additional, in the HMBC spectrum of APG showing the the cross peak from H-1” (δ = 5.15) to

C-7 (δ = 161.85) confirmed that the glucosylation takes place in the 7-OH position. Similarly,

the cross peak from H-1” (δ = 5.15) to C-7 (δ = 149.68) in the HMBC profile of BCG indicated

the glucosylation in C-7 position in the baicalein molecule [3].

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Captions

Table S1. Substrate specificity of Arabidopsis glycosyltransferase AGT.

Table S2. (A) 1H NMR profile of APG and BCG, and (B) 13C NMR profile of APG and BCG.

Legends.

Figure S1. Amino acid alignment of putative Arabidopsis GT with relative ones showing the conserved sequence PSPG in the C-terminal domain. (1), (2), (3) and (4) sequences are highly conserved in all the compared GTs.

Figure S2. Dendrogram of deduced amino acid sequence GTs displaying the phylogenetic relationship. NP_001235161.1: Glycine max ; XP_002276981.1: Vitis vinifera, ABR57234.1: Antirrhinum majus ; AAR06919.1: Stevia rebaudiana, ACB56924.1: Hieracium pilosella; BAG31949.1: Perilla frutescens, AAO42032.1: Arabidopsis thaliana, ABS83552.1: Scutellaria baicalensis; BAH19313.1: S. baicalensis, BAG31946.1: S. laeteviolacea; BAH14962.1: Torenia hybrid cultivar; AAL40272.1: Jatropha curcas; BAE48240.1: Linaria vulgaris. UGT1A3: Homo sapien; NP_066307.1: Homo sapien; AAS41089.1: Bacillus cereus; ABA42119.2: Streptomyces antibioticus

Figure S3. TLC profile of crude extraction from the cultures. (1) Control experiment without adding of substrate, (2) and (3) Crude extract of feeding experiment with apigenin and baicalein, respectively.

Figure S4. High-performance liquid chromatography (HPLC) elution profiles of the crude extracts. (A) Crude extract of apigenin - fed experiment displaying the retention time of apigenin at 9.4 min and APG at 5.2 min. (B) In the similar manner, crude extract of baicalein-fed experiment showing the peak of baicalein at 9.2 min and BCG at 5.3 min.

Figure S5. Prep-HPLC profile of crude extract.(A) apigenin-fed E. coli extract, and (B) baicalein-fed E. coliextract.

Figure S6. HPLC profile of purified flavone glucosides. (A) apigenin-7-O-β-glucoside (APG), and (B) baicalein-7-O-β-glucoside. Purity was monitored by the area percent (%).

Figure S7. LC-QTOF-ESI/MS profile. (A) apigenin-7-O-glucoside and (B) baicalein-7-O-

glucoside.

Figure S8. (A) 1H NMR spectrum of BCG, and (B) 13C NMR spectrum of BCG.

Figure S9. (A) 1H NMR spectrum of APG, (B)13C NMR spectrum of APG.

Figure S10. (A) HMBC spectrum of APG, (B) HMBC spectrum of BCG

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777879

8081828384858687

888990

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Figure S11. SDS-PAGE analysis of protein expression. Lane 1, protein marker ; Lane 2, Insoluble fraction; Lane 3: Soluble fraction. The apparent mass of protein is about 63,7 kDa.

Figure S12. Production of glucoside by the control and mutants. MAFA and MAFB, the controls

were fed with apgenin and baicalein, respectively. Similarly, MA1FA, MA2FA, MA3FA;

MA1FB, MA2FB and MA3FB: strains MA1F, MA2F and MA3F were transformed with

apigenin and baicalein, respectively. The experiments were performed in triplicate and repeated

three times. Error bars indicate standard deviations.

Figure S13. (A). Effect of different glucose concentration on the production of BCG using

MA3F strain. GL, GL1, GL2 and GL3 indicated 0, 1, 3 and 5% of glucose-supplemented TB

media using for culture. (B) Effect of different glycerol concentration on the cell growth of

MA3F strain. G, G1, G2 and G3 indicated 0.4, 1, 1.5 and 2% of glycerol-supplemented TB

media. The experiments were performed in triplicate and repeated three times. Error bars

indicate standard deviations.

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Table S1

Type of substrate Names Chemical formula Relative bioconversion rate (%)

Flavone

Apigenin 100

Baicalein 85

Luteolin 23.5

Chrysin 5.5

Flavonol

Quercetin 0

Kaempferol 0

Myricetin

0

Daidzein 0

125

Isoflavone Genistein 0

Flavanone

Naringenin 0

Hesperetin 0

Hesperidin 0

Table S2

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(A)

Apigenin-7-O-β-glucoside Baicalein-7-O-β-glucoside

Proton Chemical shift

Number of proton

Multiple Coupling constant (J)

Proton Chemical shift

Number of proton

Multiple Coupling constant (J)

3 6.83 1H s 3 7.02 1H s

6 6.44 1H d 8 6 8.60 1H s

8 6.83 1H s 8 7.07 1H s

5 12.98 1H s 5 12.57 1H s

2’,6’ 7.97 2H d 16 2’, 6’ 8.07 2H d 16

3’,5’ 6.95 2H d 16 3’, 4’, 5’ 7.59 - 7.64

3H m

1” 5.15 1H d 8 1” 5.16 1H d 8

4” 3.21 1H m

6” 3.73 1H m

(B)

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Apigenin-7-O-β-glucoside Baicalein-7-O-β-glucoside

Carbon Chemical shift Carbon Chemical shift

C-4 182.48 C-4 183.05C-2 164.72 C-2 163.94C-9 163.43 C-9 152.09C-7 161.85 C-7 149.68C-5 161.58 C-5 146.95C-4’ 157.41 C-4’ 132.55C-6 129.09 C-6 131.30C-1’ 121.49 C-1’ 131.06

C-5’ 116.47 C-5’ 130.06C-3’ 116.47 C-3’ 130.06C-2’ 105.80 C-2’ 126.86C-6’ 105.80 C-6’ 126.86C-10 103.58 C-10 106.56C-3 100.35 C-3 105.20C-1” 99.98 C-1” 101.40C-8 95.30 C-8 94.71C-3” 77.64 C-3” 77.81C-5” 76.90 C-5” 76.34C-2” 73.56 C-2” 73.65C-4” 70.00 C-4” 70.13

C-6” 61.06 C-6” 61.10

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Figure S1172

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Figure S2

Figure S3

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Figure S4

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Figure S7

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OHO

OOH

OH

OO

OOH

OOH

HOHO OH

OH

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Figure S8

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Figure S11

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Figure S12

12 h 24 h 36 h 48 h 60 h0

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MAFAMAFBMA1FAMA1FBMA2FAMA2FBMA3FAMA3FB

Incubation time (h)

Prod

uct (

mgL

-1)

Figure S13

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12 h 24 h 36 h 48 h 60 h0

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GLGL1GL3GL2

Incubation time (h)

Prod

uct (

mg/

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(B)

12 h 24 h 36 h 48 h 60 h0

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GG1G3G2

Incubation time (h)

Prod

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