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Fermentative Production of Phenolic Glucosides by Escherichia coli with an Engineered Glucosyltransferase from Rhodiola sachalinensis Qinglin He, Hua Yin, Jingjie Jiang, Yanfen Bai, Ning Chen, Shaowei Liu, Yibin Zhuang, and Tao Liu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 26 May 2017 Downloaded from http://pubs.acs.org on May 30, 2017

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Journal of Agricultural and Food Chemistry

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Fermentative Production of Phenolic Glucosides by Escherichia coli with an Engineered

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Glucosyltransferase from Rhodiola sachalinensis

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Qinglin He, †,‡ Hua Yin, ‡,§ Jingjie Jiang,⊥ Yanfen Bai, ‡,§ Ning Chen,† Shaowei Liu,⊥ Yibin

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Zhuang, *,‡,§ Tao Liu *,‡,§

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†

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College of Biotechnology，Tianjin University of Science and Technology, Tianjin 300457,

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China

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‡

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China

National and Local United Engineering Lab of Metabolic Control Fermentation Technology,

Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308,

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§

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300308, China

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⊥

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University of Science and Technology, Shanghai 200237, China

Key Laboratory of Systems Microbial Biotechnology, Chinese Academy of Sciences, Tianjin

College of Biotechnology, the State Key Laboratory of Bioreactor Engineering, East China

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ACS Paragon Plus Environment

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ABSTRACT: Three rosmarinic acid analogs produced by recombinant Escherichia coli, two

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xanthones from fungi, and honokiol from plants, were explored as the substrates of E. coli

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harboring a glucosyltransferase mutant UGT73B6FS to generate phenolic glucosides. Six new

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and two known compounds were isolated from fermentation broth of the recombinant strain of

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the feeding experiments, and the compounds were identified by spectroscopy. The

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biotransformation of rosmarinic acid analogs and xanthones into corresponding glucosides was

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presented for the first time. This study not only demonstrated the substrate flexibility of the

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glucosyltransferase mutant UGT73B6FS towards aromatic alcohols but also provided an effective

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and economical method to produce phenolic glucosides by fermentation circumventing the use of

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expensive precursor UDP-glucose.

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KEYWORDS: biotransformations, UGT73B6FS, E. coli, phenolic glucosides

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INTRODUCTION

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Phenolic compounds are among the most widespread class of metabolites in nature, and are

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almost ubiquitous in plants such as medicinal herbs, cereals, vegetables and fruits.1,2 These

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phenolic substances range from simple phenolic acids to complex flavonoids, which are often

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decorated with alkyl, sugar and other moieties.2-4 The effects of phenolic compounds as anti-aging,

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anti-inflammatory, antioxidant and anti-proliferative agents by several mechanisms, including

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the elimination of free radicals and the protection and regeneration of other dietary antioxidants,

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have been demonstrated.5,6 Phenolic compounds have played important roles in nutraceutical,

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pharmaceutical and cosmetic industries.1,2,6

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Glycosylation of natural products catalyzed by UDP-glycosyltransferases (UGTs), can often

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enhance the solubility and bioavailability of compounds, and reduce the toxicity of

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xenobiotics.7,8 Importantly, the sugar moieties often take part in interaction with biological

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targets, and thus are critical for the efficacy of compounds.9,10 Hence, glycosyltransferase has

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been developed as one of the important tools for generating natural products with improved

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physical, chemical and biological properties. A large number of UGTs have been identified from

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plants and microbial sources to modify natural products by glycosylation,11 and many of them

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have been employed for glucosylation of natural products with diverse structures.12-15 For

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example, the UGT YjiC has been shown to accept resveratrol, epothilone A, geldanamycin

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analogues, various flavonoids, etc., as substrates.13,16-18 OleD has been engineered to increase

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their flexibility in accepting various donor and acceptor substrates, and its variants have been

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revealed to glucosylate over 100 diverse acceptors such as macrolides, flavonoids,

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aminocoumarins, indolocarbozoles, polyenes, and steroids to form O-/S-/N-glucosides.14,19


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Ye et al. first isolated the glucosyltransferase UGT73B6 from Rhodiola sachalinensis, and

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found that it could convert tyrosol into salidroside.20 We achieved the synthesis of salidroside in

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a recombinant Escherichia coli (E. coli) strain containing Rhodiola-derived UGT73B6.21 We

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found that UGT73B6 could also catalyze the formation of icariside D2 by attaching glucose to

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the phenolic position of tyrosol.21 Subsequently, a mutant UGT73B6FS was generated by directed

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evolution, which improved the enzymatic activity and regioselectivity of UGT73B6 toward

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aromatic alcohols.22 The potential of UGT73B6FS as a tool to catalyze the glucosylation of

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phenolic compounds has not been fully assessed.

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In this study, we tested the substrate flexibility of UGT73B6FS toward phenols and produced

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corresponding glucosides by using an E. coli harboring UGT73B6FS. Six structurally diverse

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phenolic acceptors from different sources, including three rosmarinic acid (RA) analogs

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produced by recombinant E. coli,23,24 two xanthones from fungi,25 and honokiol from plants,

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were tested as the substrates for UGT73B6FS. Ten products were generated, of which eight

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including six new compounds, were isolated and identified by NMR. The biotransformation of

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rosmarinic acid analogs and xanthones into corresponding glucosides was presented for the first

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time. In the process, we have revealed a flexible UGT, which might be a useful tool to produce

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phenolic glycosylated products with improved biological activities.

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MATERIALS AND METHODS

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General Experimental Procedures. For the analyses of biotransformation products, 20 µL of

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fermented broth supernatants were examined by HPLC-MS analysis, using an Agilent 1260

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system (Agilent, Santa Clara, USA) with 1260 Infinity UV detector and a Bruker microQ-TOF II

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mass spectrometer (Bruker BioSpin, Switzerland) equipped with an electrospray ionization (ESI)

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interface. An Innoval C18 column (4.6 × 250 mm; i.d., 5 µm; Agela, Tianjin, China) was used


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for HPLC system. The elution conditions were as follows: solvent A = H2O containing 0.1%

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formic acid; solvent B = methanol; flow rate = 1 mL/min; 0-5 min 5% B, 6-45 min 5% B

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increased to 100% B (linear gradient); compounds 1–13 were detected at 254 nm and compounds

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14–16 were detected at 201 nm. Mass spectra were acquired in the positive ion mode. The ion

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spray was operated at 25 Arb N2/min, 3.5 kV, and 300 °C.

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NMR spectra were determined using a Bruker Avance Ⅲ 400 spectrometer (Bruker BioSpin,

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Switzerland) at 400 MHz (for 1H) and 100 MHz (for

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(ppm) and coupling constants (J) were given in hertz (Hz), calibrating the chemical shifts using

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13

C). Chemical shifts were expressed in δ

the solvent signal or TMS as reference.

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Bacterial Strains and Chemicals. The recombinant E. coli BL21 (DE3) with pET-Um

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harbouring UGT73B6FS named GAS-f2, constructed in our previous work, was used for

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biotransformation experiments, and the E. coli BL21 (DE3) with pET28a served as empty

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control.22 The DNA and protein sequences of UGT73B6FS are provided in Supporting

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Information Table S1.

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Rosmarinic acid analogues and xanthones were prepared and characterized by our laboratory

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as described previously.23-25 Honokiol was purchased from Aladdin chemistry Co., Ltd.

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(Shanghai, China). Other chemicals were purchased from Sinopharm Chemical Reagent Co., Ltd.

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(Shanghai, China). All solvents used for analyses were of HPLC grade. All other chemicals used

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were of analytical grade.

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Biotransformation Experiments. Single colonies of the recombinant E. coli BL21 (DE3)

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strains were cultured overnight in Luria-Bertani (LB) medium containing 50 µg/mL kanamycin

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at 37 °C. Then, the overnight cultures were inoculated into 50 mL fresh LB medium at 1:100 and

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incubated in a gyratory shaker incubator at 37 °C, 200 rpm. When OD600 reached 0.6-0.8,


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enzyme expression was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final

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concentration of 0.1 mM. Subsequently the cultures were shaken at 16 °C for 24 h; the cells were

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centrifuged, washed and re-suspended in 50 mL M9Y medium (M9 minimal salts, 5 mM

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MgSO4, 0.1 mM CaCl2, 2% (w/v) glucose, supplemented with 1% (w/v) yeast extract).

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Substrates for biotransformation were added into the cultures at a final concentration depending

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on the amount of individual substrates. The cultures were incubated for 48 h and the supernatants

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were analyzed by HPLC-MS.

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Extraction and Isolation of Phenolic Glucosides. After the fermentation broths were

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centrifuged, the supernatants containing biotransformation products were extracted with glass

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columns wet-packed with the macroporous resin SP825L (50 mL; Sepabeads, Kyoto, Japan),

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respectively. An aliquot (200 mL) of distilled water and 80% (v/v) ethanol was sequentially

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loaded into the column and eluted at a constant flow rate of 1 mL/min. The eluate of 80% (v/v)

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ethanol was separately condensed under reduced pressure. The residue was then dissolved in 2

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mL methanol, and purified by semi-preparative HPLC using a Shimadzu LC-6 AD with SPD-

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20A detector, equipped with a YMC-pack ODS-A column (10 × 250 mm; i.d., 5 µm; YMC,

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Kyoto, Japan). The flow rate was 4 mL/min, and other HPLC conditions were the same as

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described above. The compounds were characterized using NMR.

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Coumaryl-phenyllactate 4′-O-β-D-glucoside (4). 1H NMR (DMSO-d6, 400 MHz) δ 7.68 (2H,

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d, J = 8.8 Hz, H-2′, H-6′), 7.59 (1H, d, J = 15.9 Hz, H-7′), 7.31 (2H, m, H-2, H-6), 7.30 (2H, m,

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H-3, H-5), 7.28 (1H, m, H-4), 7.08 (2H, d, J = 8.7 Hz, H-2′, H-6′), 6.50 (1H, d, J = 15.9 Hz, H-

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8′), 4.95 (1H, d, J = 7.3 Hz, H-1′′), 5.17 (1H, dd, J = 8.8, 4.3 Hz, H-8), 3.69 (1H, d, J = 11.4 Hz,

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H-6′′a), 3.46 (1H, dd, J = 11.4, 5.6 Hz, H-6′′b), 3.36 (1H, m, H-5′′), 3.26 (1H, m, H-3′′), 3.24

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(1H, m, H-2′′), 3.17 (1H, dd, J = 14.2, 4.3 Hz, H-7a), 3.16 (1H, m, H-4′′), 3.09 (1H, dd, J = 14.2,


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4.3 Hz, H-7b); 13C NMR (DMSO-d6, 100 MHz) δ 171.2 (C, C-9), 166.3 (C, C-9′), 159.7 (C, C-

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4′), 145.3 (CH, C-7′), 137.3 (C, C-1), 130.6 (CH, C-2′, C-6′), 129.8 (CH, C-2, C-6), 128.7 (CH,

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C-3, C-5), 128.0 (C, C-4), 127.1 (CH, C-1′), 116.9 (CH, C-3′, C-5′), 115.8 (CH, C-8′), 100.4

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(CH, C-1′′), 77.6 (CH, C-5′′), 77.0 (CH, C-3′′), 73.6 (CH, C-2′′), 73.4 (CH, C-8), 70.1 (CH, C-

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4′′), 61.1 (CH2, C-6′′), 37.2 (CH2, C-7); HRESIMS m/z 497.1418 [M + Na]+ (calcd for

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C24H26O10Na, 497.1424).

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Caffeoyl-phenyllactate 4′-O-β-D-glucoside (5). 1H NMR (CD3OD, 400 MHz) δ 7.56 (1H, d, J

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= 15.9 Hz, H-7′), 7.32 (2H, m, H-2, H-6), 7.30 (2H, m, H-3, H-5), 7.23 (1H, m, H-4), 7.21 (1H, d,

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J = 8.4 Hz, H-5′), 7.11 (1H, d, J = 2.1 Hz, H-2′), 7.05 (1H, dd, J = 8.4, 2.1 Hz, H-6′), 6.36 (1H, d,

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J = 15.9 Hz, H-8′), 5.27 (1H, dd, J = 8.7, 4.1 Hz, H-8), 4.87 (1H, d, J = 7.6 Hz, H-1′′, overlap

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with signal of CD3OD), 3.92 (1H, dd, J = 12.1, 1.9 Hz, H-6′′a), 3.73 (1H, dd, J = 12.0, 5.0 Hz,

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H-6′′b), 3.51-3.47 (4H, m), 3.28 (1H, dd, J = 14.2, 4.1 Hz, H-7a), 3.16 (1H, dd, J = 14.3, 8.8 Hz,

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H-7b); 13C NMR (CD3OD, 100 MHz) δ 166. 6 (C, C-9′), 147.6 (C, C-4′), 147.1 (C, C-3′), 145.3

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(CH, C-7′), 136.8 (C, C-1), 129.6 (C, C-1′), 129.0 (CH, C-2, C-6), 128.0 (CH, C-3, C-5), 126.4

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(C, C-4), 120.9 (C, C-6′), 115.3 (CH, C-8′), 116.7 (CH, C-5′), 114.6 (CH, C-2′), 102.1 (CH, C-

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1′′), 77.0 (CH, C-5′′), 76.1 (CH, C-3′′), 73.4 (C, C-8, C-2′′), 69.9 (CH, C-4′′), 61.0 (CH2, C-6′′),

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37.2 (C, C-7), not detected (C-9); HRESIMS m/z 513.1368 [M + Na]+ (calcd for C24H26O11Na,

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513.1373).

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Feruloyl-phenyllactate 4′-O-β-D-glucoside (6). 1H NMR (CD3OD, 400 MHz) δ 7.62 (1H, d, J

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= 15.9 Hz, H-7′), 7.31 (2H, m, H-2, H-6), 7.30 (2H, m, H-3, H-5), 7.24 (2H, m, H-4, H-2′), 7.18

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(1H, dd, J = 8.4, 1.5 Hz, H-6′), 7.15 (1H, d, J = 8.2 Hz, H-5′), 6.44 (1H, d, J = 15.9 Hz, H-8′),

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5.31 (1H, dd, J = 8.6, 4.2 Hz, H-8), 4.99 (1H, d, J = 7.3 Hz, H-1′′), 3.90 (1H, dd, J = 12.1, 1.8 Hz,

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H-6′′a), 3.89 (3H, s, OCH3), 3.72 (1H, dd, J = 12.0, 5.1 Hz, H-6′′b), 3.51-3.41 (4H, m), 3.28 (1H,


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dd, J = 14.3, 4.2 Hz, H-7a), 3.18 (1H, dd, J = 14.3, 8.6 Hz, H-7b); 13C NMR (CD3OD, 100 MHz)

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δ 171.2 (C, C-9), 166.6 (C, C-9′), 149.6 (C, C-4′), 148.8 (C, C-3′), 145.5 (CH, C-7′), 136.5 (C, C-

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1), 129.0 (CH, C-2, C-6), 128.9 (C, C-1′), 128.0 (CH, C-3, C-5), 126.5 (C, C-4), 122.4 (C, C-6′),

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115.9 (CH, C-5′), 115.2 (CH, C-8′), 111.0 (CH, C-2′), 100.8 (CH, C-1′′), 76.9 (CH, C-5′′), 76.4

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(CH, C-3′′), 73.4 (CH, C-2′′), 73.1 (CH, C-8), 69.9 (CH, C-4′′), 61.1 (CH2, C-6′′), 55.4 (CH3,

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OCH3), 37.1 (CH2, C-7); HRESIMS m/z 527.1531 [M + Na]+ (calcd for C25H28O11Na, 527.1528).

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3,4,8-Trihydroxy-6-methoxy-1-methylxanthone 3, 4-O-β-D-diglucoside (8). 1H NMR (DMSO-

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d6, 400 MHz) δ 13.18 (1H, s, 8-OH), 7.13 (1H, s, H-2), 6.62 (1H, d, J = 2.3 Hz, H-5), 6.35 (1H, d,

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J = 2.3 Hz, H-7), 5.23 (1H, d, J = 4.5 Hz, Glc-OH), 5.18 (1H, d, J = 4.5 Hz, Glc-OH), 5.15 (1H,

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d, J = 4.5 Hz, Glc-OH), 5.09 (1H, d, J = 7.4 Hz, H-1′), 4.96 (1H, d, J = 4.9 Hz, Glc-OH), 4.91

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(1H, d, J = 7.6 Hz, H-1′′), 4.62 (1H, t, J = 5.6 Hz, Glc-OH), 4.40 (1H, t, J = 5.8 Hz, Glc-OH),

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3.88 (3H, s, OCH3), 3.49-3.14 (10H, m), 2.76 (3H, s, CH3); 13C NMR (DMSO-d6, 100 MHz) δ

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181.9 (C, C-9), 165.9 (C, C-6), 162.5 (C, C-8), 156.5 (C, C-10a), 153.7 (C, C-3), 150.9 (C, C-4a),

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136.4 (C, C-1), 131.8 (C, C-4), 115.2 (CH, C-2, observed from HMBC), 113.4 (C, C-9a),

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104.4/100.6 (CH, C-1′/1′′), 103.1 (C, C-8a), 96.9 (CH, C-7), 92.4 (CH, C-5), 77.2/77.1 (CH, C-

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5′/5′′), 76.2/76.0 (CH, C-3′/3′′), 74.1/73.3 (CH, C-2′/2′′), 72.3/69.6 (CH, C-4′/4′′), 62.9/60.8 (CH2,

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C-6′/6′′), 56.0 (CH3, OCH3), 22.9 (C, CH3); HRESIMS m/z 635.1563 [M + Na]+ (calcd for

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C27H33O16Na, 635.1588).

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3,4,8-Trihydroxy-6-methoxy-1-methylxanthone 3-O-β-D-glucoside (9).1H NMR (DMSO-d6,

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400 MHz) δ 13.31 (1H, s, 8-OH), 7.04 (1H, s, H-2), 6.58 (1H, d, J = 2.3 Hz, H-5), 6.33 (1H, d, J

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= 2.3 Hz, H-7), 4.96 (1H, d, J = 7.5 Hz, H-1′), 3.88 (3H, s, OCH3), 3.73 (1H, d, J = 12.0 Hz, H-

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6′a), 3.43-3.28 (5H, m), 2.70 (3H, s, CH3);

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165.8 (C, C-6), 162.7 (C, C-8), 156.6 (C, C-10a), 149.1 (C, C-3), 133.0 (C-4, observed from

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C NMR (DMSO-d6, 100 MHz) δ 182.3 (C, C-9),


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HMBC), 130.0 (C-1, observed from HMBC), 113.4 (C, C-9a), 114.7 (C-2, observed from

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HMBC), 103.0 (C, C-8a), 101.2 (CH, C-1′), 96.8 (CH, C-7), 91.9 (CH, C-5), 77.3 (CH, C-5′),

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75.8 (CH, C-3′), 73.2 (CH, C-2′), 69.6 (CH, C-4′), 62.9 (CH2, C-6′), 55.9 (CH3, OCH3), 22.6

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(CH3, CH3), not detected (C-4a); HRESIMS m/z 473.1041 [M + Na]+ (calcd for C21H22O11Na,

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473.1060).

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3,6,8-Trihydroxy-4-methoxy-1-methylxanthone 3-O-β-D-glucoside (12). 1H NMR (DMSO-d6,

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400 MHz) δ 13.40 (1H, s, 8-OH), 6.71 (1H, s, H-2), 6.65 (1H, d, J = 2.3 Hz, H-5), 6.38 (1H, d, J

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= 2.3 Hz, H-7), 5.10 (1H, d, J = 7.4 Hz, H-1′), 3.84 (3H, s, OCH3), 3.71 (dd, J = 9.9, 4.5 Hz, 1H,

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H-6′a), 3.45-3.25 (5H, m), 2.68 (3H, s, CH3); 13C NMR (DMSO-d6, 100 MHz) δ 181.7 (C, C-9),

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163.4 (C, C-6), 162.5 (C, C-8), 156.0 (C, C-3), 156.0 (C, C-10a), 151.7 (C, C-4a), 136.2 (C-1),

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132.8 (C, C-4), not detected (C-2), 111.0 (C-9a, observed from HMBC), 103.4 (C, C-8a), 101.2

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(CH, C-1′), 98.5 (CH, C-7), 94.0 (CH, C-5), 77.3 (CH, C-5′), 75.8 (CH, C-3′), 73.1 (CH, C-2′),

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69.6 (CH, C-4′), 60.6 (CH3, OCH3), 60.6 (CH2, C-6′), 22.6 (CH3, CH3); HRESIMS m/z 451.1168

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[M + H]+ (calcd for C21H23O11, 451.1240).

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Honokiol 2,4′-O-β-D-diglucoside (15). 1H NMR (CD3OD, 400 MHz) δ 7.40 (dd, J = 8.5, 2.2

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Hz, 1H, H-6′), 7.37 (d, J = 2.2 Hz, 1H, H-2′), 7.18 (d, J = 8.5 Hz, 2H, H-3, 5′), 7.08 (dd, J = 8.2,

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2.2 Hz, 1H, H-4), 7.07 (d, J = 2.2 Hz, 1H, H-6), 6.06 (m, 1H, H-8′), 5.97 (m, 1H, H-8), 5.09 (m,

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2H, H-9), 5.04 (m, 2H, H-9′), 4.98 (d, J = 7.4 Hz, 1H, H-1′′′), 4.93 (d, J = 7.4 Hz, 1H, H-1′′),

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3.91 (dd, J = 12.1, 1.8 Hz, 1H, H-6′′a), 3.86 (dd, J = 12.1, 2.1 Hz, 1H, H-6′′′a), 3.72 (dd, J = 12.0,

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5.0 Hz, 1H, H-6′′b), 3.68 (dd, J = 12.0, 5.2 Hz, 1H, H-6′′′b), 3.51–3.35 (12H, overlap); 13C NMR

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(CD3OD, 100 MHz) δ154.4 (C-4′), 152.4 (C-2), 137.7 (C-8), 137.3 (C-8′), 133.9 (C-5), 132.6 (C-

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1), 131.1 (C-1′), 130.9 (C-2′), 130.4 (C-6), 129.0 (C-3′), 128.4 (C-6′), 127.9 (C-4), 115.3 (C-3),

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114.6 (C-5′), 114.4 (C-9′), 114.3 (C-9), 101.4/100.6 (C-1′′, 1′′′), 76.9 (C-3″/3′″), 76.8/76.7 (C-


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5′′/5′′′), 73.7/73.6 (C-2′′/ 2′′′), 70.0/69.9 (C-4′′/4′′′), 61.2/61.1 (C-6′′/6′′′), 39.0 (C-7), 33.9 (C-7′);

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HRESIMS m/z 613.2158 [M + Na]+ (calcd for C30H38O12Na, 613.2261).

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RESULTS AND DISCUSSION

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Glucosylation of Rosmarinic Acid Analogs in Recombinant E. coli. The phenolic

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compound RA and its derivatives such as lithospermic acid, salvianolic acid, and yunnaneic acid

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have diverse biological activities, including anti-oxidant, anti-inflammatory, anti-tumor, and

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anti-microbial properties.26 Hence, RA and its derivatives have been studied for large-scale

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production as dietary or pharmaceutical supplements to improve human health.27 As we know,

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several glucosylated derivatives of RA and analogs have been isolated from plants, and the

216

position and the number of glycosyl groups present in the molecule plays a significant role in the

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antioxidant activity.28 Thus, RA and analogs are interesting targets for studying glucosylation by

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biocatalysis.

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Previously, we utilized enzymatic actions of At4CL and CbRAS to investigate the production

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of various RA analogs in E. coli by precursor-directed biosynthesis, and obtained some RA

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derivatives.23,24 We chose three high yield products from the above experiments, coumaryl-

222

phenyllactate (1), caffeoyl-phenyllactate (2), feruloyl-phenyllactate (3) as substrates for

223

glycosylation. A total of 15 mg of these substrates were independently fed to the fermentation

224

broth of the recombinant E. coli expressing UGT73B6FS. After fermentation, the metabolites

225

were subjected to HPLC-MS analyses, and three new peaks (products 4, 5 and 6) were observed.

226

The biotransformation products were extracted and purified by resin SP825L and semi-

227

preparative HPLC. Products 4 (19 mg, yield 83%), 5 (12 mg, yield 54%), and 6 (5 mg, yield

228

22%) were suggested to be monoglucosides according to HRESIMS peaks at m/z 475.1592,

229

491.1552, 505.1711 [M + H]+, and 497.1418, 513.1368, 527.1531 [M + Na]+, respectively. 1H


10

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230

and 13C NMR data were analyzed to determine the structures of biotransformation products 4, 5,

231

and 6. The glucose moieties were further confirmed at the C-4′ position by HMBC correlations

232

between the anomeric proton and the C-4′ carbon. The coupling constants (J = 7.2 Hz for 4 and

233

6, J = 7.6 Hz for 5) between the H-1′′ glucose and H-2′′ indicated that the glucose molecules

234

were in the β-configuration. Thus, the structures of biotransformation products 4, 5, and 6 were

235

confirmed as coumaryl-phenyllactate 4′-O-β-D-glucoside, caffeoyl-phenyllactate 4′-O-β-D-

236

glucoside, and feruloyl-phenyllactate 4′-O-β-D-glucoside, respectively (Figure 1).

237

Glucosylation of Xanthones in Recombinant E. coli. Xanthones are an important class of

238

natural products, and are commonly occurred in higher plant families, fungi, and lichen.29 Their

239

structures are related to those of flavonoids, and are classified into six major groups, including

240

simple xanthones, xanthone glycosides, prenylated xanthones, xanthonolignoids, bisxanthones,

241

and miscellaneous xanthones. Xanthones exhibit a broad spectrum of biological activities, such

242

as hepatoprotective, antimicrobial, anti-carcinogenic, anti-leprosy, antioxidant, anticholinergic

243

and mutagenicity.30-33 In contrast, xanthone-O-glycosides were observed to have central nervous

244

system depressant effect in mice and rats.34 Xanthones have become increasingly important in

245

drug discovery because of their medicinal properties.

246

Our previous chemical investigation of the secondary metabolites of fungus Penicillium sp.

247

NH-7-1 resulted in the isolation of five xanthones.25 Of these, 3,4,8-Trihydroxy-6-methoxy-1-

248

methylxanthone (7, 8 mg) and 3,6,8-trihydroxy-4-methoxy-1-methylxanthone (11, 5 mg) were

249

selected for preparing the biotransformation. When compound 7 was incubated with E. coli

250

overexpressing UGT73b6FS, three new glucosylated products were generated, as seen by HPLC

251

analysis (Figure 2). The number of glucose moieties attached to the substrate was determined

252

using HPLC-MS analysis. Two biotransformation products were purified by semi-preparative


11


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253

HPLC for NMR spectra, while the third one was not isolated due to low yield. Eventually, the

254

structures of products 8 (1.8 mg, yield 10.6%) and 9 (1.1 mg, yield 8.8%) were elucidated based

255

on extensive NMR data analyses. The NMR data of 8 showed typical chemical shifts for a

256

skeleton of 7 plus a set of signals corresponding to two glucose units. In the HMBC data of 8, the

257

HMBC correlations of H-1′ (δH 5.09, d, J = 7.4 Hz) with C-3 (δC 153.7), and H-1′′ (δH 4.91, d, J

258

= 7.6 Hz) with C-4 (δC 131.8) suggested two β-glucosyl moieties attached at 3-OH and 4-OH of

259

7, respectively. Product 8 was thus determined to be 3,4,8-trihydroxy-6-methoxy-1-

260

methylxanthone 3, 4-O-β-D-diglucoside. Product 9 exhibited similar spectroscopic data as 7,

261

except for a substitution at 3-OH. In the HMBC data of product 9, the correlation between H-1′

262

(δH 4.96, d, J = 7.5 Hz) with C-3 (δC 149.1) suggested a β-glucosyl moiety positioned at 3-OH of

263

7. Therefore, product 9 was identified as 3,4,8-trihydroxy-6-methoxy-1-methylxanthone 3-O-β-

264

D-glucoside. According to HPLC-MS analysis, the molecular formula of product 10 was

265

established as C21H22O11 based on HRESIMS data m/z 451.1149 [M + H]+ and 473.1046 [M +

266

Na]+. The molecular weight indicated the compound 10 is a monoglucoside with the glucose

267

moiety attached to a hydroxyl position different from compound 9 with a glucose linked to 3-

268

position. The structure of product 8 with 3, 4 diglucosides indicated that the glucosyltransferase

269

catalyzed transferring glucose moiety to both 3 and 4-OH of 7. Thus, we assumed that

270

glucosylation occurred on 4-OH in the formation of 8.

271

When substrate 11 (5 mg) was incubated with recombinant E. coli strain harboring

272

UGT73B6FS, two products were observed by HPLC analysis (Figure 3). The HPLC-MS

273

analyses confirmed that the compounds contained one glucose moiety. The glucosides of

274

xanthone were purified by semi-preparative HPLC. One of the products, compound 12 (2.4 mg,

275

yield 30.7%), obtained in sufficient quantities, was further characterized by NMR. The HMBC


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276

correlation of the H-1′ (δH 5.10) with C-1 (δC 163.4) established a link between the glucose and

277

6-hydroxy group of xanthone (11). The chemical shift values and the coupling constants (J = 7.4

278

Hz) of the anomeric protons indicated that the glucose molecule was also in the β-configuration.

279

Thus, the structure of product 12 was confirmed as 6-O-β-D-glucoside. According to HPLC-MS

280

analysis, the molecular formula of product 13 was established as C21H22O11 based on HRESIMS

281

data m/z 451.1169 [M + H]+. The molecular weight indicated the compound 13 is a

282

monoglucoside with the glucose moiety attached to a hydroxyl position different from compound

283

12 with a glucose moiety being linked to 6-position. In above experiments, the

284

glucosyltransferase catalyzed transferring glucose moiety to both 3 and 4-OH of 7. As the 4-OH

285

group of compound 11 was decorated with a methyl group, the glucose moiety was most likely

286

connected to 3-OH in compound 12.

287

The xanthone glucosides described here have not been reported from a natural source.

288

Intriguingly, the glucosyltransferase UGT73B6FS attached two glucose moieties to the xanthone

289

compound in positions 3 and 4. Though the biological activities of these compounds have not yet

290

been determined, they might have some medicinal, cosmetic, and pharmacological properties, as

291

those previously identified.33,34

292

Glucosylation of Honokiol in Recombinant E. coli. Honokiol (14), a bioactive biphenyl-

293

neolignan isolated from the Chinese herb Magnolia officinalis, is widely used as a tribal remedy

294

for gastrointestinal disorders, cough, anxiety and allergies.35 Honokiol has a wide range of

295

biological activities, such as anti-oxidative, anti-fungal, anticancer, antiviral and anti-

296

inflammatory.36 Although structural modifications and total synthesis of honokiol have been

297

investigated, the synthesis of honokiol O-glucosides by biotransformation in microorganisms has

298

been attempted using UGTs from fungi, and only monoglucoside products were obtained.37


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299

In the present study, honokiol (26 mg) was added as the sugar acceptor in the fermentation

300

broth of the recombinant E. coli strain. After incubation, the reaction product was analyzed by

301

HPLC-MS. As shown in Figure 4, two peaks were observed and the molecular mass ion of the

302

major peak (15) at 37.5 min was 591.2321 [M + H]+, suggesting that the molecular mass

303

increased by 324 as compared to that of honokiol. It indicated that two glucose molecules were

304

attached to honokiol. Products 15 (23 mg, yield 39.9%) and 16 (3 mg, yield 7.2%) were purified

305

by semi-preparative HPLC for NMR analysis. The 1D and 2D NMR spectra of product 15

306

measured in CD3OD also showed signals of honokiol and two glucose units. The HMBC

307

correlations of the H-1′′ (δH 4.93) with C-4′ (δC 154.4), and H-1′′′ (δH 4.98) with C-2 (δC 152.4)

308

established a link between the glucose moieties and hydroxy groups of honokiol. The chemical

309

shift values and the coupling constants (J = 7.4 Hz) of the anomeric protons indicated that both

310

glycosidations were β-linkages. Hence, this product was identified as honokiol 2, 4′-O-β-D-

311

diglucoside.

312

The molecular mass ion of the minor peak (16) at 41.2 min was 451.1585 [M + Na]+

313

according to HPLC-MS analysis, 162 more than that of honokiol, which implied that one

314

molecule of glucose was attached to substrate honokiol. The structure of 16 was confirmed as

315

honokiol 4′-O-β-D-glucoside by 1H and 13C NMR data, which were consistent with previously

316

reported characterization data.37

317

The results obtained in our experiments showed that E. coli harboring UGT73B6FS could

318

glucosylate phenolic compounds, including simple phenolic compounds,21,22 coumarin 4-

319

methylumbelliferone,22 RA analogs, xanthones, and honokiol, into their glucosides. In this study,

320

six compounds including RA analogs and xanthone glucosides have not been previously

321

reported. Intriguingly, glucosyltransferase catalyzes the formation of xanthone and honokiol with


14

Page 15 of 26


322

two glucoses in different positions. The exact sequence of the glucosylation event of 3, 4

323

position of xanthone (compound 8) need to be further investigated in the future. Based on

324

products formed by biotransformation experiments, the glucosylation of honokiol most likely

325

occurred first in 4′ position.

326

Phenolic acid including RA and derivatives is widely used in the fields of functional food and

327

medicine.26,38 Previously, we generated 18 RA analogs by precursor-directed biosynthesis.23,24

328

All these compounds can also be tested as the substrates of UGT73B6FS in the future. Those

329

studies demonstrate the potential of combinatorial biosynthesis to synthesize phenolic acid

330

analogs by harnessing acetyltransferases and glycosyltransferases. Many phenolic compounds

331

including phenolic acids and xanthones can be potential targets for glucosylation by the

332

glycosyltransferase. The glucosylation of phenolic compounds may improve bioavailability,

333

stability and biological activities, providing lead compounds for nutraceutical and

334

pharmaceutical industry. The new glucosylated products will also provide the possibility to

335

investigate the changes in biological functions caused by glucosylation. The work demonstrated

336

the substrate flexibility of the glucosyltransferase mutant UGT73B6FS towards aromatic alcohols

337

and provided a new means for the molecular structure modification of natural lead compounds.

338

ASSOCIATED CONTENT

339

Supporting Information

340

This supporting information is available free of charge via the Internet at http://pubs.acs.org. 1H

341

and 13C NMR spectra of 4–6, 8, 9, 12, and NMR data of 16.

342

AUTHOR INFORMATION

343

Corresponding Author

344

*Phone: +86-22-24828718. E-mail: [email protected]; [email protected].


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345

Funding

346

This work was supported by grants from the National Natural Science Foundation of China (no.

347

21302214), the 973 Program of China (no. 2012CB721100), and the Biological Resources

348

Service Plan of CAS (no. ZSTH-023).

349

Notes

350

The authors declare no competing financial interest.

351

ACKNOWLEDGEMENTS

352

REFERENCES

353

(1) Cheynier, V. Phenolic compounds: from plants to foods. Phytochem. Rev. 2012, 11, 153–177.

354

(2) Pereira, D. M.; Valentão, P.; Pereira, J.A.; Andrade, P. B. Phenolics: From chemistry to

355

biology. Molecules 2009, 14, 2202–2211.

356

(3) Babbar, N.; Oberoi, H. S.; Sandhu, S. K.; Bhargav, V. K. Influence of different solvents in

357

extraction of phenolic compounds from vegetable residues and their evaluation as natural sources

358

of antioxidants. J. Food Sci. Technol. 2014, 51, 2568–2575.

359

(4) Heim, K. E.; Tagliaferro, A. R.; Bobilya, D. J. Flavonoid antioxidants: Chemistry,

360

metabolism and structure-activity relationships. J. Nutr. Biochem. 2002, 13, 572–584.

361

(5) Rong, T. Chemistry and biochemistry of dietary polyphenols. Nutrients 2010, 2, 1231–1246.

362

(6) Lin, D. R.; Xiao M. S.; Zhao, J. J.; Li, Z. H.; Xing, B. S.; Li, X. D.; Kong, M. Z.; Li, L. Y.;

363

Zhang, Q.; Liu, Y. W.; Chen, H.; Qin, W.; Wu, H. J.; Chen, S. Y. An overview of plant phenolic

364

compounds and their importance in human nutrition and management of type 2 diabetes.

365

Molecules 2016, 21, 1374: 1–19.

366

(7) Woo, H. J.; Kang, H. K.; Nguyen, T. T. H.; Kim, G. E.; Kim, Y. M.; Park, J. S.; Kim, D.;

367

Cha, J.; Moon, Y. H.; Nam, S. H.; Xia, Y. M.; Kimura, A.; Kim, D. Synthesis and


16

Page 17 of 26


368

characterization of ampelopsin glucosides using dextransucrase from Leuconostoc mesenteroides

369

B-1299CB4: glucosylation enhancing physicochemical properties. Enzyme Microb. Technol.

370

2012, 51, 311–318.

371

(8) Raab, T.; Barron, D.; Arce Vera, F.; Crespy, V.; Oliveira, M.; Williamson, G. Catechin

372

glucosides: occurrence, synthesis, and stability. J. Agric. Food Chem. 2010, 58, 2138–2149.

373

(9) Weymouth-Wilson, A. C. The role of carbohydrates in biologically active natural products.

374

Nat. Prod. Rep. 1997, 14, 99–110.

375

(10) Griffith, B. R.; Langenhan, J. M., Thorson, J. S. ‘Sweetening’ natural products via

376

glycorandomization. Curr. Opin. Biotechnol. 2005, 16, 622–630.

377

(11) Blanchardn, S.; Thorson, J. S. Enzymatic tools for engineering natural product

378

glycosylation. Curr. Opin. Chem. Biol. 2006, 10, 263–271.

379

(12) Xie, K. B.; Chen, R. D.; Li, J. H; Wang, R. S.; Chen, D. W.; Dou, X. X.; Dai, J. G.

380

Exploring the catalytic promiscuity of a new glycosyltransferase from Carthamus tinctorius.

381

Org. Lett. 2014, 16, 4874–4877.

382

(13) Pandey, R. P.; Gurung R. B.; Parajuli, P.; Koirala, N.; Tuoi L. T.; Sohng, J. K. Assessing

383

acceptor substrate promiscuity of YjiC-mediated glycosylation toward flavonoids. Carbohydr.

384

Res. 2014, 393, 26–31.

385

(14) Zhou, M. Q. Hamza, A.; Zhan, C. G.; Thorson, J. S. Assessing the regioselectivity of OleD-

386

catalyzed glycosylation with a diverse set of acceptors. J. Nat. Prod. 2013, 76, 279–286.

387

(15) Weis, M.; Lim, E. K.; Bruce, N.; Bowles, D. Regioselective glucosylation of aromatic

388

compounds: screening of a recombinant glycosyltransferase library to identify biocatalysts.

389

Angew. Chem. Int. Ed. 2006, 45, 3534–3538.


17


Page 18 of 26

390

(16) Pandey, R. P.; Parajuli, P.; Shin, J. Y.; Lee, J.; Lee, S.; Hong, Y. S.; Park, Y. I., Kim, J. S.;

391

Sohng, J. K. Enzymatic biosynthesis of novel resveratrol glucoside and glycoside derivatives.

392

Appl. Environ. Microbiol. 2014, 80, 7235–7243.

393

(17) Parajuli, P.; Pandey, R. P.; Koirala, N.; Yoon, Y. J.; Kim, B. G.; Sohng, J. K. Enzymatic

394

synthesis of epothilone A glycosides. AMB Express 2014, 4, 31:1–10.

395

(18) Huo, Q.; Li, H. M.; Lee, J. K.; Li, J.; Ma, T.; Zhang, X.; Dai, Y.; Hong, Y. S.; Wu, C. Z.

396

Biosynthesis of novel glucosides geldanamycin analogs by enzymatic synthesis. J. Microbiol.

397

Biotechnol. 2016, 26, 56–60.

398

(19) Williams, G. J.; Gantt, R, W.; Thorson, J. S. The impact of enzyme engineering upon

399

natural product glycodiversification. Curr. Opin. Chem. Biol. 2008, 12, 556–564.

400

(20) Ma, L. Q.; Liu, B. Y.; Gao, D. Y.; Pang, X. B.; Lü, S. Y.; Yu, H. S.; Wang, H.; Yan, F.; Li,

401

Z. Q.; Li, Y. F.;, Ye, H. C. Molecular cloning and overexpression of a novel UDP-

402

glucosyltransferase elevating salidroside levels in Rhodiola sachalinensis. Plant Cell Rep. 2007,

403

26, 989–999.

404

(21) Bai, Y. F.; Bi, H. P.; Zhuang, Y. B.; Liu, C.; Cai, T.; Liu, X. N., Zhang, X. L.; Liu, T.; Ma,

405

Y. H. Production of salidroside in metabolically engineered Escherichia coli. Sci. Rep. 2014, 4,

406

6640: 1–8.

407

(22) Bai, Y. F.; Yin, H.; Bi, H. P.; Zhuang, Y. B.; Liu, T.; Ma, Y. H. De novo biosynthesis of

408

gastrodin in Escherichia coli. Metab. Eng. 2016, 35, 138–147.

409

(23) Jiang, J. J.; Bi, H. P.; Zhuang, Y. B.; Liu, S. W.; Liu, T.; Ma, Y. H. Engineered synthesis of

410

rosmarinic acid

411

caffeoylphenyllactate. Biotechnol. Lett. 2016, 38, 81–88.

in

Escherichia

coli

resulting production

of a

new intermediate,


18

Page 19 of 26


412

(24) Zhuang, Y. B.; Jiang, J. J.; Bi, H. P.; Yin, H.; Liu, S. W.; Liu, T. Synthesis of rosmarinic

413

acid analogues in Escherichia coli. Biotechnol. Lett. 2016, 38, 619–627.

414

(25) Zhuang, Y. B.; Yin, H.; Zhang, X. W.; Zhou, W.; Liu, T. Three new xanthones from the

415

fungus Penicillium sp. NH-7-1. Helv. Chim. Acta 2015, 98, 699–703.

416

(26) Bulgakov, V. P.; Inyushkina, Y. V.; Fedoreyev, S. A. Rosmarinic acid and its derivatives:

417

biotechnology and applications. Crit. Rev. Biotechnol. 2012, 32, 203–217.

418

(27) Bloch, S. E.; Schmidt-Dannert C. Construction of a chimeric biosynthetic pathway for the

419

de novo biosynthesis of rosmarinic acid in Escherichia coli. ChemBioChem. 2014, 15, 2393–

420

2401.

421

(28) Le, C. E.; Schwaiger, S.; Banaigs, B.; Stuppner, H.; Gafner, F. Distribution of a new

422

rosmarinic acid derivative in Eryngium alpinum L. and other Apiaceae. J. Agric. Food Chem.

423

2005, 53, 4367–4372.

424

(29) Cardona M. L.; Fernandez, I.; Pedro, J. R.; Serrano, A. Xanthones from Hypericum

425

reflexum. Phytochemistry 1990, 29, 3003–3006.

426

(30) Jiang, Y.; Zhang, W.; Tu, P. F.; Xu, X. J. Xanthone glycosides from Polygala tenuifolia and

427

their conformational analyses. J. Nat. Prod. 2005, 68, 875–879.

428

(31) Negi, J. S.; Singh, P.; Rawat. B. Chemical constituents and biological importance of

429

Swertia: a review. Curr. Res. Chem. 2011, 3, 1–15.

430

(32) Peres, V.; Nagem, T. J.; de Oliveira, F. F. Tetraoxygenated naturally occurring xanthones.

431

Phytochemistry 2000, 55, 683–710.

432

(33) Negi, J. S.; Bisht, V. K.; Singh, P.; Rawat, M. S. M.; Joshi, G. P. Naturally occurring

433

xanthones: chemistry and biology. J. appl. Chem. 2013, 2013, 1–9.

434

(34) Ghosal, S.; Sharma, P. V.; Chaudhuri, R. K. Chemical constituents of gentianaceae X:


19


Page 20 of 26

435

xanthone-O-glucosides of Swertia purpurascens wall. J. Pharm. Sci. 1974, 63, 1286–1290.

436

(35) Kumar, A.; Kumar，S. U. Chaudhary, A. Honokiol analogs: a novel class of anticancer

437

agents targeting cell signaling pathways and other bioactivities. Future Med. Chem. 2013, 5,

438

809-829.

439

(36) Katiyar, S. K. Emerging phytochemicals for the prevention and treatment of head and neck

440

cancer. Molecules 2016, 21, 1610: 1–13.

441

(37) Yang, L.; Wang, Z. H.; Lei, H.; Chen, R. D.; Wang, X. L.; Peng, Y.; Dai, J. G.

442

Neuroprotective glucosides of magnolol and honokiol from microbial-specific glycosylation.

443

Tetrahedron 2014, 70, 8244–8251.

444

(38) Kim, G. D.; Park, Y. S.; Jin, Y. H.; Park, C. S. Production and applications of rosmarinic

445

acid and structurally related compounds. Appl. Microbiol. Biotechnol. 2015, 99, 2083–2092.

446 447 448 449 450 451 452 453 454 455 456 457


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FIGURE CAPTIONS

459

Figure 1. (A) Biotransformation process for the biosynthesis of rosmarinic acid analogs

460

glucosides; (B), (C), and (D) HPLC-UV chromatograms of rosmarinic acid analogs and their

461

glucosides in the fermentation supernatants of recombinant strains (Black lines indicate controls

462

and blue lines indicate biotransformation samples); (E), (F), and (G) MS analyses of products 4,

463

5, and 6, respectively.

464

Figure 2. (A) Biotransformation process for the biosynthesis of 3,4,8-trihydroxy-6-methoxy-1-

465

methylxanthone glucosides; (B) HPLC-UV chromatograms of 3,4,8-trihydroxy-6-methoxy-1-

466

methylxanthone and its glucosides in the fermentation supernatant of recombinant strains (Black

467

line indicates control, blue line indicates biotransformation sample); (C), (D), and (E) MS

468

analyses of products 8, 9, and 10, respectively.

469

Figure 3. (A) Biotransformation process for the biosynthesis of 3,6,8-trihydroxy-4-methoxy-1-

470

methylxanthone glucosides; (B) HPLC-UV chromatograms of 3,6,8-trihydroxy-4-methoxy-1-

471

methylxanthone and its glucosides in the fermentation supernatant of recombinant strains (Black

472

line indicates control, blue line indicates biotransformation sample); (C) and (D) MS analyses of

473

products 12 and 13, respectively.

474

Figure 4. (A) Biotransformation process for the biosynthesis of honokiol glucosides; (B) HPLC-

475

UV chromatograms of honokiol and its glucosides in the fermentation supernatant of

476

recombinant strains (Black line indicates control, blue line indicates biotransformation sample);

477

(C) and (D) MS analyses of products 15 and 16, respectively.

478 479 480


21


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Figure 1.

482 483 484 485 486 487 488 489 490 491 492


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Figure 2.

494 495 496 497 498 499 500 501 502 503 504 505


23


506

Page 24 of 26

Figure 3.

507 508 509 510 511 512 513 514 515 516 517 518 519 520 521 522


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Figure 4.

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526

527

528

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Fermentative Production of Phenolic Glucosides by ... - ACS Publications

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