One-pot Multi-enzyme Cofactors Recycling (OPME-CR) System for

Subscriber access provided by - Access paid by the | UCSB Libraries

Bioactive Constituents, Metabolites, and Functions

One-pot Multi-enzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production Sumangala Darsandhari, Ramesh Prasad Pandey, Biplav Shrestha, Prakash Parajuli, Kwangkyoung Liou, and Jae Kyung Sohng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02421 • Publication Date (Web): 03 Jul 2018 Downloaded from http://pubs.acs.org on July 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 29

Journal of Agricultural and Food Chemistry

Journal of Agricultural and Food Chemistry One-pot Multi-enzyme Cofactors Recycling (OPME-CR) System for Lactose and Non-natural Saccharide Conjugated Polyphenol Production Sumangala Darsandhari1,Ϯ, Ramesh Prasad Pandey1,2,Ϯ, Biplav Shrestha1, Prakash Parajuli1, Kwangkyoung Liou1,2, Jae Kyung Sohng1,2,* 1

Department of Life Science and Biochemical Engineering, SunMoon University, 70 Sunmoon-ro 221, Tangjeong-

myeon, Asan-si, Chungnam 31460, Republic of Korea 2

Department of BT-Convergent Pharmaceutical Engineering, SunMoon University, 70 Sunmoon-ro 221,

Tangjeong-myeon, Asan-si, Chungnam 31460, Republic of Korea.

Ϯ

These authors contributed equally.

*Corresponding author: Prof. Jae Kyung Sohng Tel: +82(41)530-2246 Fax: +82(41)530-8229 Email: [email protected]

1 ACS Paragon Plus Environment


1

ABSTRACT

2

A one-pot multi-enzyme cofactors recycling (OPME-CR) system was designed for the synthesis of UDP-α-D-

3

galactose which was combined with LgtB, a β-(1,4) galactosyltransferase from Neisseria meningitidis to modify

4

various polyphenol glycosides. This system recycles one mole of ADP and one mole of UDP to regenerate one mole

5

of UDP-α-D-galactose by consuming two moles of acetylphosphate and one mole of D-galactose in each cycle. The

6

ATP additionally used to generate UDP from UMP was also recycled at the beginning of the reaction. The

7

engineered cofactors recycling system with LgtB efficiently added a D-galactose unit to a variety of sugar units such

8

as D-glucose, rutinose, and 2-deoxy-D-glucose. The temperature, pH, incubation time, and divalent metal ions for

9

the OPME-CR system were optimized. The maximum number of UDP-α-D-galactose regeneration cycles (RCmax)

10

was 18.24 by fed batch reaction. The engineered system generated natural and non-natural polyphenol saccharides

11

efficiently and cost-effectively.

12 13

Keywords. Lactoside derivatives, Glycosylation, One pot system, Cofactors recycling

14


Page 2 of 29

Page 3 of 29


15

INTRODUCTION

16

Sugars are structural components frequently found in a large number of natural products. Important drugs such as

17

amphotericin B, avermectins, vancomycin, erythromycin, and doxorubicin, contain sugars appended to their

18

aglycone structure.1 These sugars participate in the biomolecular recognition of the cellular target by a bioactive

19

compound; their existence is therefore essential for the biological activity of many natural products.2 The vast

20

majority of the natural products (NPs) produced by microbes are naturally glycosylated and many of them are

21

utilized as drugs or drug leads owing to their biological properties.1,3 Plant secondary metabolites are also decorated

22

with different sugar units, which play distinct physicochemical and biological roles in plant physiology, interspecies

23

interactions,4 and human and animal health.5 A number of plant and animal metabolites are coated with different

24

sugars and they have their own functions. One of the metabolites of Gaultheria yunnanensis (FRANCH) REHDER

25

(G yunnanensis), methylsalicyclate 2-O-β-D-lactoside, demonstrates a potential therapeutic role, for example, in the

26

treatment of rheumatoid arthritis, swelling, and pain.6 Similarly, multifunctional human milk oligosaccharides such

27

as 3′-sialyllactose, 6′-sialyllactose, and brain gangliosides (GD1, GT1, GQ1) also carry lactose as part of their

28

structures.7,8 The sugar unit plays a vital and wide-ranging role such as in immune system, bacterial and viral

29

infections.9,10

30

Chemo-enzymatic approaches are often employed for the synthesis of diverse types of glycoconjugates.11,12 Such

31

methods are more efficient and less expensive than the chemical methods entailing harsh chemical treatment and

32

several protection/deprotection steps before any product can be obtained.13 Glycosyltransferases (GT) catalyze the

33

formation of regio- and stereo-specific glycosidic linkages between specific sugar donors and aglycones.14-16 LgtB is

34

a β-1,4-galactosyltransferase gene from Neisseria meningitidis,

35

galactose to the terminal lacto-N-neotetraose via a β-1,4 linkage.17 LgtB also has been reported to transfer D-

36

galactose

37

galactosyltransferase/-UDP-4'-gal-epimerase fusion protein was prepared to carry out two sequential steps of an

38

important biosynthetic galactosylation pathway for the synthesis of galactosylated oligosaccharides.19 β-1,4-

39

galactosyltransferase has been used to incorporate a D-galactose unit at the terminal N-acetylglucosamine or D-

40

glucose in glycoproteins, glycolipids or other compounds (eg. carbohydrates, epothiolone, and vancomycin) with β-

41

linkage for the biosynthesis of lipo-oligolysaccharides (or their lactosylated derivatives).20-22 Recently our group

42

synthesized quercetin 3-O-β-D-lactoside and it is found to have a better anticancer activity than quercetin 3-O-β-D-

to

the

4′-OH

position

of

D-glucose

and

which transfers D-galactose from UDP-α-D-

N-acetylglucosamine.18


Previously,

the

β-1,4-


43

glucoside.23 As far as to our knowledge, previous work about the production of other polyphenol saccharides

44

carrying lactose moiety has not been reported yet.

45

In this study, we designed a one-pot multi-enzyme cofactor recycling (OPME-CR) system for the continuous

46

regeneration of UDP-α-D-galactose re-using D-glucose-1-phosphate, UDP, and ADP in the reaction system

47

(Scheme 1), resulting in cost-effective synthesis of the derivatives of various polyphenol saccharides carrying a D-

48

galactose moiety. Thus, using the OPME-CR system, we conjugated the D-galactose moiety into various

49

polyphenolic glycosides that harbor sugars such as D-glucose, rutinose, and 2-deoxy-D-glucose in their structure.

50

One of the substrates, quercetin 3-O-β-D-glucoside, was used as a model compound and the reaction conditions

51

were optimized. Newly synthesized quercetin 3-O-β-D-lactoside was characterized by various nuclear magnetic

52

resonance (NMR) studies. The products of other substrates were characterized by ultrahigh performance liquid

53

chromatography-photo diode array (UHPLC-PDA) and high-resolution quadruple time-of-flight electrospray

54

ionization mass spectrometry (HR-QTOF-ESI/MS) analyses.

55

MATERIALS AND METHODS

56

Culture media and chemicals

57

All the compounds used in this study were either purchased from Sigma–Aldrich (St. Louis, MO, USA) or were

58

available in the laboratory library. Standard quercetin 3-O-β-D-glucoside, and DMSO-d6 were purchased from

59

Sigma-Aldrich Inc. (USA). α-D-glucose-1-phosphate, UDP-α-D-galactose, isopropyl-β-D-thiogalactopyranoside

60

(IPTG), adenine triphosphate (ATP), uridine monophosphate (UMP), and acetylphosphate were purchased from

61

GeneChem (Daejeon, South Korea). All the other chemicals and reagents used were purchased from high grade

62

commercial sources. Other substrates like α-mangostein 3-O-β-D-glucoside,24 resveratrol 3-O-β-D-glucoside,25

63

curcumin 4′-O-β-D-glucoside,26 biochanin A 7-O-β-D- glucoside, formononetin 7-O-β-D- glucoside,27 emodin 3-O-

64

β-D-glucoside, and aloe emodin 3-O-β-D-glucoside,28 used in this study were available in the laboratory.

65

Recombinant enzymes expression and purification

66

The expression of proteins, acetate kinase (ACK-pET24ma, GenBank accession: WP_000095711.1), UMP kinase

67

(UMK-pET15b, GenBank accession: WP_001483307.1), α-D-glucose-1-phosphate uridylyltransferase (GalU-

68

pET24ma, GenBank accession: AIF36551.1), galactokinase (GalK, GenBank accession: YP_489030.1) and

69

galactose-1-phosphate uridylyltransferase (GalT, GenBank accession: BAA35420.1) were performed as described

70

previously.29,30 Further, the gene β-1,4-galactosyltransferase (LgtB, GenBank: AAM33872.1) was synthesized from


Page 4 of 29

Page 5 of 29


71

GenScript (USA) with restriction sites NdeI and HindIII and cloned into pET24ma. The expression of all the genes

72

was performed in the host Escherichia coli BL21 (DE3) (Stratagene, USA). All the recombinant strains were grown

73

using respective seed cultures supplemented with the antibiotic (50 µg/mL kanamycin or 100 µg/mL ampicillin) in

74

Luria–Bertani (LB) media and incubated at 37°C until the optical density at 600 nm (OD600nm) reached ~0.8.

75

Subsequently, the cells were induced by adding IPTG to a final concentration of 0.5 mM via incubation at 20°C for

76

approximately 20 h. The cells were harvested by centrifugation at 842 x g for 15 min at 4°C and washed twice with

77

a buffer (50 mM Tris-HCl of pH 7.5, 100 mM NaCl and 10% glycerol). The cells were sonicated, and the clear

78

lysate was collected by high-speed centrifugation at 13,475 x g for 30 min at 4°C. The proteins were purified using

79

TALON metal nickel affinity resin (Takara Bio, Shiga, Japan). The resin was equilibrated by washing with a buffer

80

containing 100 mM Tris-HCl (pH 7.5) and 300 mM NaCl before adding a crude lysate of soluble proteins. The

81

mixture of protein and resin was gently agitated in ice for 30 min and eluted with various concentrations (10, 100,

82

200, and 500 mM) of imidazole. The fractions were examined using 12% sodium dodecyl sulfate-polyacrylamide

83

gel electrophoresis (SDS-PAGE). The concentrations of the enzymes were determined using Bradford’s method.

84

Regular galactosylation using LgtB enzyme

85

The regular galactosylation reaction was conducted in a 500 µL volume containing a 50 mM Tris-HCl buffer (pH

86

7.5), 10 mM MgCl2, 2 mM quercetin 3-O-β-D-glucoside, 50 µg/mL purified LgtB enzyme, and 10 mM UDP-α-D-

87

galactose. The mixture was incubated at 37°C for 3 h, followed by quenching the reaction with 400 µL chilled

88

methanol. It was vortexed, mixed well, filtered through a 0.2 µm filter and subjected to UHPLC-PDA and HR-

89

QTOF-ESI/MS analyses.

90

Galactosylation reaction using various concentration of UDP-α-D-galactose and quercetin 3-O-β-D-glucoside

91

The various concentrations of quercetin-3-O-β-D-glucoside (3 mM, 5 mM, and 10 mM) and UDP-α-D-galactose (3

92

mM, 10 mM, and 20 mM) were used for the reaction at 37°C with 50 mM Tris-HCl buffer (pH 7.5) using reaction

93

ingredients similar to the regular galactosylation reaction mixture. The 2 mM quercetin-3-O-β-D-glucoside was

94

maintained constant at different concentrations of the donor, while 10 mM UDP-α-D-galactose was held constant at

95

the different substrate concentrations.

96

Optimization of one-pot multi-enzyme cofactors recycling (OPME-CR) galactosylation reaction

97

To continuously regenerate the UDP-α-D-galactose, a donor substrate for the LgtB galactosylation reaction, an

98

OPME system was engineered using a combination of six recombinant enzymes of different origins. The UDP-α-D-



99

galactose regeneration system required continuous use of two molecules of ATP cofactors. Thus, the separate

100

system of ATP regeneration was coupled with the UDP-α-D-galactose regeneration system. The OPME

101

galactosylation reaction mixture included 50 mM Tris-HCl buffer (pH 7.5), 10 mM MgCl2, 70 mM acetylphosphate,

102

20 mM D-galactose, 1 mM α-D-glucose-1-phosphate, 0.25 mM ATP, 0.25 mM UMP, and 2 mM quercetin 3-O-β-D-

103

glucoside along with an additional six purified enzymes: UMK (50 µg/mL), ACK (50 µg/mL), GalU (50 µg/mL),

104

GalK (50 µg/mL), GalT (50 µg/mL) and LgtB (50 µg/mL). The final reaction volume was 500 µL.

105

Identical OPME reactions were carried out to optimize the temperature of the galactosylation reaction in different

106

vials and incubated at different temperatures (20°C, 30°C, 37°C, and 45°C) for 3 h. A 10 µL of the reaction sample

107

was drawn at different time intervals and quenched with 490 µL chilled methanol. The samples were analyzed using

108

UHPLC-PDA.

109

To study the effect of pH on the galactosyltransferase activity of LgtB enzyme, identical reaction mixtures with 3

110

mM quercetin 3-O-β-D-glucoside were prepared in a 50 mM of different buffers (carbonate-bicarbonate buffer,

111

citrate buffer, phosphate buffer, Tris-HCl buffer and glycine buffer) at various pH (pH 4 to pH 10.5) and incubated

112

at 37°C for 3 h. The sample preparation method was similar to the method described above.

113

For a preparative scale reaction, a 20 mL reaction volume containing quercetin 3-O-β-D-glucoside was treated under

114

the optimized conditions of temperature (37°C), pH (8), and divalent metal ions (Mg2+). The starting concentration

115

of quercetin 3-O-β-D-glucoside was 2 mM. After 0.5 h of the reaction, 1 mM quercetin 3-O-β-D-glucoside was

116

added to the reaction mixture. Similarly, additional quercetin 3-O-β-D-glucoside was added during 1.5 h and 2.5 h

117

of the reaction. The pH of the reaction mixture was maintained throughout the reaction with 0.1 M NaOH. Other

118

reaction ingredients used were 70 mM acetylphosphate, 20 mM D-galactose, 10 mM Mg2+, 1 mM α-D-glucose-1-

119

phosphate, 0.25 mM ATP, 0.25 mM UMP, and six purified enzymes as mentioned above. The progress of the

120

reaction was monitored by UHPLC-PDA for up to 12 h. The maximum number regeneration cycles (RCmax) of

121

UDP-α-D-galactose was calculated as the total amount of quercetin 3-O-β-D-lactoside (mM) produced per 0.25 mM

122

of UMP.

123

OPME-CR galactosylation reaction with other polyphenolic glycosides

124

After optimizing the conditions, the OPME reaction mixture (500 µL) for the attachment of a D-galactose unit to

125

various compounds was prepared by using 50 mM glycine buffer (pH 8), 10 mM MgCl2, 70 mM acetylphosphate,

126

20 mM D-galactose, 1 mM α-D-glucose-1-phosphate, 0.25 mM ATP, 0.25 mM UMP, and 2 mM substrate


Page 6 of 29

Page 7 of 29


127

(kaempferol 3-O-β-D-glucoside, biochanin A 7-O-β-D-glucoside, formononetin 7-O-β-D-glucoside, resveratrol 3-O-

128

β-D-glucoside, curcumin 4′-O-β-D-glucoside, emodin 3-O-β-D-glucoside, aloe emodin 3-O-β-D-glucoside, rutin,

129

diosmin, and α-mangostein 3-O-β-2-deoxy-D-glucoside), and the purified enzymes (UMK, ACK, GalU, GalK, GalT,

130

and LgtB) at similar concentrations as described above.

131

The reactions were incubated at 37°C for 2 h, and reaction samples were extracted at different time intervals and

132

quenched by adding a double volume of chilled methanol and vortexed. After centrifugation at 13,475 x g for 5 min

133

and filtering with a 0.2 µm filter, the supernatants of the reaction mixture were analyzed by UHPLC-PDA and LC-

134

QTOF-ESI/MS in positive ion mode. Appropriate dilutions were made as needed for the UHPLC-PDA analysis.

135

Analytical methods

136

The samples were analyzed using a Thermo Scientific Dionex Ultimate 3000 UHPLC-PDA system consisting of

137

High Pressure Gradient Rapid Separation HPG-3200RS series pumps, a Thermo Scientific™ Dionex™ UltiMate™

138

ACC-3000 autosampler, and a UV-Vis absorbance diode array detector, operated using Thermo Scientific™

139

Dionex™ Chromeleon™ 7.2 Chromatography Data System (CDS) software. The reverse-phase UHPLC-PDA

140

analysis was performed using a C18 column (Mightysil RP-18 GP (4.6 mm × 250 mm, 5-µm particle size) (Kanto

141

Chemical, Japan) with HPLC-grade water containing 0.05% trifluoroacetic acid (TFA) (A) and acetonitrile (B) from

142

Mallinckrodt Baker (Phillipsburg, NJ, USA) at a flow rate of 1 mL/min for 25 min. The elution protocol was as

143

follows: starting with 10% B, a linear gradient from 10 to 30% B for 0−5 min, 30−50% B for 5−10 min, 50−90% B

144

for 10−15 min, 90−70% B for 15−18 min, 70−10% B for 18−25 min was performed, followed by washing and

145

equilibration of the column. Absorbance of the eluent was monitored from 200 nm to 600 nm. The maximum UV

146

absorbance of each compound was used to monitor the chromatograms. Calibration curves of the standard quercetin

147

3-O-β-D-glucoside and the purified quercetin 3-O-β-D-lactoside were prepared for the quantification of quercetin 3-

148

O-β-D-lactoside, whereas conversion percentage of other polyphenols was determined using integrated peak area of

149

substrate and product in HPLC chromatogram.

150

The HR-QTOF ESI/MS analysis was performed using an ACQUITY (UPLC, Waters Corp., Billerica, MA, USA)

151

column coupled with a SYNAPT G2-S (Water Corp.) and the separation was performed with water (A) and

152

acetonitrile (B) as mobile phases. The column temperature was set to 35°C, the injection volume was 5 µL, and the

153

flow rate was 0.3 mL/min. The total analysis time per sample was 12 min. The gradient program was as follows: 0–

154

7 min, 0–100% B; 7–9.5 min, 100% B; 9.5–12 min, 0% B. The conditions for HR-QTOF ESI/MS measurement



Page 8 of 29

155

were

156

600 L/h; capillary voltage, 3 kV; cone voltage, 40 V; source temperature, 120°C. Collision energy of the MS was set

157

4eV and the mass spectra were recorded from m/z 50 to 1500. Data acquisition was managed by the MassLynx

158

software.

159

The purification of quercetin 3-O-β-D-lactoside was performed using preparative HPLC equipped with a C18 column

160

(YMC-Pack ODS-AQ) (250×20 mm I.D., 10 µm) connected to a UV detector (SPD-20A) with a 40 min binary

161

program. The percentage of acetonitrile used was as follows: 10−40% (0−15 min), 40−90% (15−30 min), and 90−10%

162

(30−40 min) at a flow rate of 10 mL/min. The solvent of collected sample was evaporated. The product was

163

lyophilized to remove water molecules and dissolved in DMSO-d6 for nuclear magnetic resonance (NMR) analysis

164

using a Bruker-BioSpin Avance 700 MHz NMR Spectrometer (Germany) using a Cryogenic TCi probe (5 mm) for

165

elucidation of the structure. One-dimensional NMR (1H-NMR,

166

(heteronuclear single quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC)) were

167

performed to elucidate the structure of the quercetin 3-O-β-D-lactoside.

168

RESULTS

169

Expression and preparation of enzymes

170

The expressions of individual recombinant proteins were examined using SDS-PAGE. The protein marker was used

171

to verify the size of the proteins by 12% SDS-PAGE analysis which was in good agreement with those of the

172

calculated values obtained for each protein such as LgtB (~32 kDa), UMK (~26 kDa), ACK (~40 kDa), GalU (~38

173

kDa), GalK (~35 kDa), and GalT (~35 kDa) (Fig. S1). The soluble fractions of the enzymes were purified and used

174

for the reactions.

175

Galactosylation of quercetin 3-O-β-D-glucoside

176

Galactosylation by LgtB enzyme was verified using the reaction mixture containing UDP-α-D-galactose as the sugar

177

donor and quercetin 3-O-β-D-glucoside as the acceptor substrate as described in the Methods. UHPLC-PDA of this

178

reaction mixture yielded a product peak at a retention time (tR) of ~ 7.8 min (Fig. 1A). The UV spectra of the

179

substrate and product were similar (Fig. 1B). The product peak was further analyzed by LC-QTOF-ESI/MS. In the

180

positive-ion mode, a mass fragment of m/z+ 627.1569 was found, which matched with [quercetin 3-O-β-D-lactoside

181

+ H]+, the exact calculated mass of which was 627.1561 Da (Fig. 1C). In addition, other product ions of the

182

synthesized compound were analyzed to confirm the presence of sugar units (Fig. 1D). A distinct fragment ion of

as

follows:

ionization

mode,

positive; desolvation temperature,

13


300°C;

desolvation

gas

flow,

C-NMR) and two-dimensional NMR

Page 9 of 29


183

the disaccharide sugar unit was found (m/z+ 325.1132) along with other mass spectra characterizing fragments of

184

quercetin 3-O-β-D-glucoside and quercetin aglycone.

185

Structural analysis

186

The compound dissolved in NMR grade DMSO-d6 was analyzed using 1H, 13C, HSQC, and HMBC-NMR analyses.

187

The 1H-NMR spectrum of the product showed two anomeric protons with doublets at δ 5.49 (J=7.769 Hz) and δ

188

4.21 (J=7.2 Hz), representing the beta (β) configuration of the two sugar moieties, whereas other proton signals for

189

sugar moieties (D-glucose and D-galactose) were observed in the region from δ (3.0–4.0) ppm (Table 1; Fig. S2).

190

The anomeric carbons of D-glucose and D-galactose were obtained at δ 101.22 ppm and δ 104.29 ppm, respectively

191

(Table 2; Fig. S3), and their correlations were measured in HSQC for confirmation of the position of sugar

192

conjugation (Fig. S4). The correlation between 1″-H at δ 5.49 ppm and the 3rd position carbon of quercetin (C-3) at δ

193

133.56 ppm confirmed the conjugation of D-glucose moiety at the 3-OH position of quercetin Fig. S5. The

194

correlation between 1′′′-H of δ 4.21 ppm and 4″-C at δ 80.94 (4th carbon of glucose moiety) confirmed the

195

conjugation of D-galactose via (1-4) β-linkage obtained from the HMBC (Figs. S4 and S5). The 1H and 13C-NMR

196

data of quercetin 3-O-β-D-glucoside, and quercetin 3-O-β-D-lactoside are compared in Tables 1 and 2.14,31 The

197

spectra obtained here for lactose moiety are consistent with the previously published results. 20,22

198

Reaction using various concentration of UDP-α-D-galactose and quercetin 3-O-β-D-glucoside

199

The conversion percentage of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside was observed at varying

200

concentrations of UDP-α-D-galactose (3 mM, 10 mM, and 20 mM) while keeping the concentration of quercetin 3-

201

O-β-D-glucoside constant (Fig. 2). Similarly, quercetin 3-O-β-D-glucoside concentration was varied from 3 mM to

202

10 mM while keeping the concentration of UDP-α-D-galactose constant in an another set of reactions. When 3 mM

203

of the donor UDP-α-D-galactose was used, only 42% of the 2 mM quercetin 3-O-β-D-glucoside was converted to

204

the product (0.84 mM) in 2 h. After increasing the UDP-α-D-galactose concentration to 10 mM, almost all the

205

substrate was converted into the product (~1.99 mM). When 20 mM UDP-α-D-galactose was used, all the substrate

206

was converted to the product within an hour of the reaction. Similarly, the maximum amount of quercetin 3-O-β-D-

207

glucoside that was galactosylated by LgtB was 3 mM in the presence of 10 mM UDP-α-D-galactose as a donor. At 5

208

mM concentration of quercetin 3-O-β-D-glucoside, the conversion was 39% (quercetin 3-O-β-D-lactoside, 1.95 mM)

209

in the first hour of reaction, which increased slightly to 42% (2.1 mM) in 2 h. Similarly, the conversion was also



210

limited to 30% (quercetin 3-O-β-D-lactoside, 3 mM) when10 mM quercetin 3-O-β-D-glucoside was used as initial

211

substrate concentration (Fig. 2).

212

Development of one-pot multi-enzyme cofactors recycling (OPME-CR) system

213

A one-pot reaction system was engineered for the continuous production of the activated nucleotide sugar donor,

214

UDP-α-D-galactose, in a single vessel while regenerating ATP cofactor using simple and inexpensive starting

215

materials such as UMP, acetylphosphate, and D-galactose (Scheme 1). The use of NDP-sugars for in vitro

216

production of a large quantity of NP glycosides at an industrial scale is limited because of their high cost and lack of

217

commercial availability. In the proposed system, the final ATP and α-D-glucose 1-phosphate concentrations

218

required are very low (1 mM each) as both are regenerated economically. The reaction system converts UMP to

219

UDP (Scheme 1) at the expense of a molecule of ATP catalyzed by a UMP kinase enzyme (UMK). Similarly, D-

220

galactose is converted to α-D-galactose-1-phosphate by a galactokinase enzyme (GalK). The byproduct ADP is

221

utilized by ACK and converted to ATP by consuming a molecule of acetylphosphate. Thus, the galactose 1-

222

phosphate generated along with the UDP-α-D-glucose in the reaction by GalU is utilized by GalT for the synthesis

223

of UDP-α-D-galactose. The LgtB enzyme uses UDP-α-D-galactose produced in the pathway to attach D-galactose to

224

the various substrates containing D-glucose, 2-deoxy-D-glucose, or rutinose. The byproduct UDP, which is

225

produced in the galactosylation reaction, is re-utilized to yield UTP at the expense of one molecule of

226

acetylphosphate catalyzed by ACK in the system. Overall, UDP is recycled whereas D-galactose and

227

acetylphosphate are consumed in this OPME-CR system to generate a continuous supply of UDP-α-D-galactose

228

required for LgtB activity as shown in Scheme 1.

229

Optimization of the OPME-CR system

230

The effect of pH, divalent cofactors, incubation temperature, and incubation time on the reaction process was

231

observed in the conversion of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside. Initially, the OPME-CR

232

reactions were carried out at four different temperatures: 20°C, 30°C, 37°C, and 45°C. At both 20°C and 30°C, the

233

concentration of the product increased with the incubation time to its maximum amount in 3h. The highest

234

conversion of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside was observed at 37°C within 2 h. Almost

235

all (99%) of the 2 mM quercetin 3-O-β-D-glucoside added to the reaction mixture was converted to a product (1.99

236

mM) at this temperature (Fig. 3A). At a higher temperature of 45°C, 81% conversion (1.62 mM) was achieved in 30


Page 10 of 29

Page 11 of 29


237

min. The prolonged incubation of the reaction mixture at this temperature resulted in a slight reduction in the

238

amount of product to 79% (1.58 mM) within 3 h.

239

Similarly, the divalent metal ion co-factor requirement for the LgtB enzyme was determined with various divalent

240

cations. Among the nine different divalent cations tested at a 10 mM concentration, Cu2+, Fe2+, and Zn2+ rendered

241

LgtB virtually inactive. As shown in fig. 3B, the enzyme also showed limited activity with Co2+ and Ni2+. LgtB

242

showed a conversion of 48% of quercetin 3-O-β-D-glucoside with Pb2+, which was higher than the level attained

243

with Ca2+. With Ca2+, 35% of quercetin 3-O-β-D-glucoside was converted to quercetin 3-O-β-D-lactoside. The

244

enzyme activity with Pb2+ was almost half compared with that of Mg2+ or Mn2+. Mg2+ and Mn2+ played equal roles

245

in activating the conversion of nearly all of the substrate into product (Fig. 3B).

246

A pH profile of the OPME-CR galactosylation reactions of quercetin 3-O-β-D-glucoside measured in carbonate-

247

bicarbonate, citrate, phosphate, Tris-HCl and glycine buffers within their appropriate pH ranges revealed that its

248

catalytic activity was optimum at pH 7.5 (Tris-HCl buffer and phosphate buffer) to pH 8 (glycine buffer) (Fig. 3C).

249

The 15% conversion (0.45 mM) observed at the lowest pH tested (pH 4) in citrate buffer increased to 96% (2.88

250

mM) at pH 8 in glycine buffer. Similar conversion was recorded in phosphate buffer (pH 7.5). A significant decrease

251

to less than 60% molar conversion of quercetin 3-O-β-D-glucoside was observed when the pH of the reaction

252

mixture was slightly basic (pH 9.0). Further lowering the pH to 10.5 produced only 30% of product (0.9 mM).

253

Preparative scale production of quercetin 3-O-β-D-lactoside using OPME-CR system

254

To obtain a maximum yield of galactosylated product from the engineered OPME-CR system, the reaction was

255

performed on a preparative scale of 20 mL reaction volume starting with 2 mM quercetin 3-O-β-D-glucoside,

256

followed by the addition of 1 mM quercetin 3-O-β-D-glucoside three times up to 2.5 h of reaction. The progress of

257

the reaction was observed for up to 12 h. 2 mM of quercetin 3-O-β-D-glucoside completely converted into product

258

in 0.5 h. In 1 h, 2.5 mM of quercetin 3-O-β-D-lactoside was produced. The titer of quercetin 3-O-β-D-lactoside

259

increased to 3.9 mM in 3 h. Similarly, the product yield calculated at different time points indicated that the

260

maximum concentration of quercetin 3-O-β-D-lactoside produced was 4.56 mM (2854.6 mg/L) in 4 h (Fig. 4). The

261

result highlights the capacity of one-pot reaction for the high-yield production of the galactosylated product.

262

Biosynthesis of polyphenol lactosides and non-natural saccharides using OPME-CR system

263

Three different sugars (D-glucose, rutinose, or 2-deoxy-D-glucose) conjugated plant secondary metabolites such as

264

flavonol, flavone, isoflavonoid, stilbene, curcuminoid, anthraquinone, and xanthonoid glycosides were used in the



265

OPME-CR galactosylation reaction (Fig. 5A). Among the 11 compounds used in the reaction, eight contained D-

266

glucose, two carried rutinose, and one harbored 2-deoxy-D-glucose in their structure. The UHPLC-PDA analysis of

267

the reaction mixtures displayed different turnover rates for each substrate. Analysis of the reaction mixtures using

268

UHPLC-PDA method along with HRQTOF–ESI/MS revealed the conjugation of D-galactose residue in all the

269

compounds tested (Figs. S6-S15). The conversion percentage of seven of the compounds tested reached greater than

270

90% after 120 min (Fig. 5B). The OPME-CR system yielded 1239 mg/L of quercetin 3-O-β-D-lactoside, 1198 mg/L

271

of kaempferol 3-O-β-D-lactoside, 1129 mg/L of aloe emodin 3-O-β-D-lactoside, 1188 mg/L of emodin 3-O-β-D-

272

lactoside, 1544 mg/L of rutin 4′′-O-β-D-galactoside, 1357 mg/L of curcumin 4′-O-β-D-lactoside, and 1422 mg/L of

273

mangostein 3-O-β-D-galactosyl (1-4) β-D-2-deoxyglucoside. The production of other compounds was significantly

274

low (Table S1): 85 mg/L of biochanin A 7-O-β-D-lactoside, 201 mg/L formononetin 7-O-β-D-lactoside, 99 mg/L of

275

resveratrol 3-O-β-D-lactoside, and 123 mg/L of diosmin 4′′-O-β-D-galactoside.

276

DISCUSSION

277

Despite the diverse physiological and pharmacological activities of natural polyphenols,32,33 their use as drugs or

278

food additives has been limited by their low water solubility and absorption. Glycosylation enhances the

279

bioavailability and pharmacological properties of compounds by increasing their solubility and stability.34,35 The

280

sugar moieties of the glycosides often participate in the recognition of their specific biological targets and enhance

281

their efficacy in drug development.1,36 Furthermore, natural product glycosides derived from plants are considered

282

pro-drugs with enhanced absorption and metabolism in the human body.37 Studies have been carried out for the

283

synthesis of D-galactose-conjugated oligosaccharides and flavonoids considering the importance of D-galactose

284

moieties by enzymatic as well as chemo-enzymatic methods.38,39 D-galactose is one of the major building blocks of

285

human milk oligosaccharides (HMOs). Recently, a galactose–aspirin covalent complex pro-drug was synthesized

286

chemically, and galactose-conjugated aspirin was found to enhance the inhibitory activity against the proliferation of

287

cancer cells.40

288

Previously, the OPME reaction was designed for the production of UDP-α-D-glucose and TDP-α-D-2-deoxy-D-

289

glucose.24-26 In this study, we designed an OPME-CR system for the production of an important nucleotide sugar,

290

UDP-α-D-galactose. This system can be used to attach UDP-α-D-glucose or UDP-α-D-galactose to various

291

substrates with a suitable GT enzyme. The GT used in this study, LgtB, is selective for the sole attachment of D-


Page 12 of 29

Page 13 of 29


292

galactose from UDP-α-D-galactose.19 Hence, we exploited its specificity for extension of various sugar-containing

293

substrates with D-galactose moiety.

294

The OPME-CR system regenerates ATP and UDP-α-D-galactose, the latter of which is coupled with LgtB-mediated

295

galactosylation of various secondary metabolite glycosides. In this system, two components, D-galactose and

296

acetylphosphate, are continuously consumed throughout the reaction process, while acetate and Pi are the

297

byproducts. The concentration of UMP and ATP used initially is also very low. Both of these substrates, which are

298

consumed in the reaction, are inexpensive and easily available, resulting in very cost-effective and sustainable

299

outcome. However, the disadvantage of the system is the simultaneous decrease in pH of the reaction because of the

300

continuous production of acetate.24 Thus, the pH should be monitored frequently in a large-scale reaction.

301

The optimal pH of the enzyme was 7.5 to 8 which is within the pH range of majority of glycosyltransferase.14, 17-20

302

Although Tris-HCl buffer has been known to retain the potential to interact with the molecules under the study, it is

303

being used frequently in several biological studies.41 In our study of buffer effect, glycine buffer and phosphate

304

buffer resulted in more than 90% conversion of quercetin 3-O-β-D-glucoside to quercetin 3-O-β-D-lactoside

305

whereas the reaction in Tris-HCl buffer resulted in reduction in product formation. Tris-HCl buffer has also been

306

found to have an inhibitory effect on the activity of glycosyltransferase from Citrus paradisi.42,43

307

The optimized OPME system for the conjugation of D-galactose residue was used to successfully convert eight

308

compounds containing D-glucose, two carrying rutinose disaccharide, and one with 2-deoxy-D-glucose. Some of the

309

substrates were completely converted to products while conversion of other substrates was significantly low. The

310

ability of the enzyme LgtB to contribute D-galactose residue to compounds containing glucoside, rutinoside, or 2-

311

deoxy-D-glucoside may be used to conjugate various oligosaccharide residues and synthesize novel glycoside

312

derivatives. In this experiment, LgtB conjugated D-galactose to various compounds within a short reaction time of 2

313

h. We successfully synthesized kaempferol 3-O-β-D-lactoside, quercetin 3-O-β-D-lactoside, biochanin A 7-O-β-D-

314

lactoside, formononetin 7-O-β-D-lactoside, resveratrol 3-O-β-D-lactoside, curcumin 4′-O-β-D-lactoside, emodin 3-

315

O-β-D-lactoside, aloe emodin 3-O-β-D-lactoside, rutin 4′′-O-β-D galactoside, diosmin 4′′-O-β-D galactoside, and α-

316

mangostein 3-O-β-D-galactosyl (1-4) β-D-2-deoxy-D-glucoside in varying quantities (Table S1). To the best of our

317

knowledge, none of these compounds has been reported from any natural source nor have they been synthesized.

318

The biological activities of these compounds may find application in a number of medicinal, cosmetic, and



319

pharmacologically active products. The potential health benefits of these compounds facilitate the development of

320

therapeutic interventions in the future safely, and cost-effectively.

321

Based on the use of engineered system, the final titer of quercetin 3-O-β-D-lactoside was 2854.6 mg/L, where the

322

maximum number of UDP-α-D-galactose regeneration cycles (RCmax) was 18.24 (4.56/0.25) by fed-batch reaction.

323

The LgtB activity was improved by the presence of large amounts of UDP-α-D-galactose. Thus, the one-pot system

324

that produces donor continuously plays a useful role. In the near future, the system will be further expanded for

325

sustainable and cost-effective production of various non-natural NDP-sugars coupled with glycosylation reactions to

326

generate a diverse array of natural product glycosides.

327

ACKNOWLEDGMENT

328

This work was supported by a grant from the Next-Generation BioGreen 21 Program (SSAC, grant#: PJ013137),

329

Rural Development Administration, Republic of Korea.

330 331

Supporting Information Available

332

Expression of recombinant proteins is shown in Figure S1; Figures (S2-S5) show the NMR of quercetin 3-O-β-D-

333

lactoside; Figures (S6-S15) show the UHPLC-PDA chromatograms of the galactosylation reaction of various

334

substrates with LgtB enzyme.

335 336

Conflict of interest

337

The authors declare that they have no competing interests.

338

Ethical approval

339

This article does not contain any studies with human participants or animals performed by any of the authors.


Page 14 of 29

Page 15 of 29

340 341


REFERENCES 1.

342 343

bacterial natural products. Chem Soc Rev. 2015, 44, 7591–7697. 2.

344 345

3.

4.

Simmonds, M. S. Flavonoid–insect interactions: recent advances in our knowledge. Phytochemistry. 2003, 64, 21–30.

5.

350 351

Griffith, B. R.; Langenhan, J. M.; Thorson. J. S. ‘Sweetening’natural products via glycorandomization. Curr Opin Biotechnol. 2005, 16, 622–630.

348 349

Weymouth-Wilson, A. C. The role of carbohydrates in biologically active natural products. Nat Prod Rep. 1997, 14, 99–110.

346 347

Elshahawi, S. I.; Shaaban, K. A; Kharel, M. K.; Thorson, J. S. A comprehensive review of glycosylated

Cheynier, V.; Comte, G.; Davies, K. M.; Lattanzio, V.; Martens, S. Plant phenolics: recent advances on their biosynthesis, genetics, and ecophysiology. Plant Physiol Biochem. 2013, 72, 1–20.

6.

Zhang, T.; Sun, L.; Liu, R.; Zhang, D.; Lan, X.; Huang, C.; Xin, W.; Wang, C.; Zhang, D.; Du, G. A novel

352

naturally occurring salicylic acid analogue acts as an anti-inflammatory agent by inhibiting nuclear factor-

353

kappa B activity in RAW264. 7 macrophages. Mol Pharm. 2012, 9, 671–677.

354

7.

Pandey, R. P.; Kim, D. H.; Woo, J.; Song, J.; Jang, S. H.; Kim, J. B.; Cheong, K. M.; Oh, J. S.; Sohng, J.

355

K. Broad-spectrum neutralization of avian influenza viruses by sialylated human milk oligosaccharides: in

356

vivo assessment of 3′-sialyllactose against H9N2 in chickens. Sci Rep. 2018, 8, 2563.

357

8.

358

Yu, R. K.; Tsai, Y. T.; Ariga, T.; Yanagisawa, M. Structures, biosynthesis, and functions of gangliosides—An overview. J Oleo Sci. 2011, 60, 537–544.

359

9.

360

10. Schnaar, R. L. Gangliosides of the vertebrate nervous system. J Mol Biol. 2016,428, 3325–3336.

361

11. Pandey, R. P.; Parajuli, P.; Koffas, M. A.; Sohng, J. K. Microbial production of natural and non-natural

Kolter, T. Ganglioside biochemistry. ISRN Biochem. 2012, 506160.

362

flavonoids: Pathway engineering, directed evolution and systems/synthetic biology. Biotechnol Adv. 2016, 34,

363

634–662.

364

12. Xiao, Z.; Guo, Y.; Liu, Y.; Li, L.; Zhang, Q.; Wen, L.; Wang, X.; Kondengaden, S. M.; Wu, Z.; Zhou, J.; Cao,

365

X. Chemoenzymatic synthesis of a library of human milk oligosaccharides. J Org Chem. 2016, 81, 5851–5865.

366

13. Muthana, S.; Cao, H.; Chen, X. Recent progress in chemical and chemoenzymatic synthesis of

367

carbohydrates. Curr Opin Chem Biol. 2009, 13, 573–581.



Page 16 of 29

368

14. Lim, E. K.; Ashford, D. A.; Hou, B.; Jackson, R. G.; Bowles, D. J. Arabidopsis glycosyltransferases as

369

biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides. Biotechnol Bioeng.

370

2004, 87, 623-631.

371 372 373 374 375

15. Coutinho, P. M.; Deleury, E.; Davies, G. J.; Henrissat, B. An evolving hierarchical family classification for glycosyltransferases. J Mol Biol. 2003, 328, 307–317. 16. Liang, D. M; Liu, J. H.; Wu, H.; Wang, B. B.; Zhu, H. J.; Qiao, J. J. Glycosyltransferases: mechanisms and applications in natural product development. Chem Soc Rev. 2015, 44, 8350–8374. 17. Wakarchuk, W. W.; Cunningham, A.; Watson, D. C.; Young, N. M. Role of paired basic residues in the

376

expression

377

meningitidis. Protein Eng. 1998, 11, 295–302.

378 379

of

active

recombinant

galactosyltransferases

from

the

bacterial

pathogen

Neisseria

18. Park, J. E.; Lee, K. Y.; Do, S. I.; Lee, S. S. Expression and characterization of β-1, 4-galactosyltransferase from Neisseria meningitidis and Neisseria gonorrhoeae. J Biochem Mol Biol. 2002, 35, 330–336.

380

19. Blixt, O.; Brown, J.; Schur, M. J; Wakarchuk, W.; Paulson, J. C.; Efficient preparation of natural and synthetic

381

galactosides with a recombinant β-1, 4-galactosyltransferase-/UDP-4′-gal epimerase fusion protein. J Org

382

Chem. 2001, 66, 2442–2448.

383 384

20. Oh, T.; Kim, D. H.; Kang, S. Y.; Yamaguchi, T.; Sohng, J. K. Enzymatic synthesis of vancomycin derivatives using galactosyltransferase and sialyltransferase. J Antibiot. 2011, 64, 103–109.

385

21. Yu, H.; Li, Y.; Zeng, J.; Thon, V; Nguyen, D. M.; Ly, T.; Kuang, H. Y.; Ngo, A.; Chen, X. Sequential one-pot

386

multienzyme chemoenzymatic synthesis of glycosphingolipid glycans. J Org Chem. 2016, 8, 10809–10824.

387

22. Parajuli, P.; Pandey, R. P.; Gurung, R. B.; Shin, J. Y.; Jung, H. J.; Kim, D. H.; Sohng, J. K. Enzymatic

388 389 390

synthesis of lactosylated and sialylated derivatives of epothilone A. Glycoconj J. 2016, 33, 137–146. 23. Darsandhari, S.; Bae, J. Y.; Shrestha, B.; Yamaguchi, T.; Jung, H. J.; Han, J. M.; Rha, C. S.; Pandey, R. P.; Sohng, J. K. Enzymatic synthesis of novel quercetin sialyllactoside derivatives. Nat Prod Res. 2018, 1-9.

391

24. Le, T. T.; Pandey, R. P.; Gurung, R. B.; Dhakal, D.; Sohng, J. K. Efficient enzymatic systems for synthesis of

392

novel α-mangostin glycosides exhibiting antibacterial activity against Gram-positive bacteria. Appl Microbiol

393

Biotechnol. 2014, 98, 8527–8538.

394 395

25. Shin, J. Y.; Pandey, R. P.; Jung, H. Y.; Chu, L. L.; Park, Y. I.; Sohng, J. K. In vitro single-vessel enzymatic synthesis of novel Resvera-A glucosides. Carbohydr Res. 2016, 424, 8–14.


Page 17 of 29


396

26. Gurung, R. B.; Gong, S. Y.; Dhakal, D.; Le, T. T.; Jung, N. R.; Jung, H. J.; Oh, T. J.; Sohng, J. K. Synthesis of

397

curcumin glycosides with enhanced anti-cancer properties using one-pot multi-enzyme glycosylation

398

technique. J Microbiol Biotechnol. 2017, 27, 1639-48.

399 400 401 402 403 404 405 406 407 408

27. Pandey, R. P.; Parajuli, P.; Koirala, N.; Lee, J. H.; Park, Y. I.; Sohng, J. K. Glucosylation of isoflavonoids in engineered Escherichia coli. Mol Cells. 2014. 37, 172. 28. Ghimire, G. P.; Koirala, N.; Pandey, R. P.; Jung, H. J; Sohng, J. K. Modification of emodin and aloe-emodin by glycosylation in engineered Escherihia coli. World J Microbiol Biotechnol. 2015. 31, 611-619. 29. Lee, J. H.; Chung, S. W.; Lee, H. J.; Jang, K. S.; Lee, S. G.; Kim, B. G. Optimization of the enzymatic one pot reaction for the synthesis of uridine 5′-diphosphogalactose. Bioprocess Biosyst Eng. 2010, 33, 71-78. 30. Lee, H. C.; Lee, S. D.; Sohng, J. K.; Liou, K. One-pot enzymatic synthesis of UDP-D-glucose from UMP and glucose-1-phosphate using an ATP regeneration system. J Biochem Mol Biol. 2004, 37, 503–506. 31. Parajuli, P.; Pandey, R. P; Sohng, J. K. Regiospecific biosynthesis of tamarixetin derivatives in Escherichia coli. J Biol Eng. 2018, 133, 113-121.

409

32. Daglia, M. Polyphenols as antimicrobial agents. Curr Opin Biotechnol. 2012, 23, 174–181.

410

33. Quideau, S.; Deffieux, D.; Douat‐Casassus, C.; Pouysegu, L. Plant Polyphenols: Chemical Properties,

411 412 413 414 415 416 417 418 419

Biological Activities, and Synthesis. Angew Chem Int Ed Engl. 2011, 50, 586–621. 34. Shimoda, K.; Otsuka, T.; Morimoto, Y.; Hamada, H.; Hamada, H. Glycosylation and malonylation of quercetin, epicatechin, and catechin by cultured plant cells. Chem Lett. 2007, 36, 1292–1293. 35. Pandey, R. P.; Sohng, J. K. Glycosyltransferase-mediated exchange of rare microbial sugars with natural products. Front Microbiol. 2016, 7, 1849. 36. Křen, V.; Řezanka, T. Sweet antibiotics–the role of glycosidic residues in antibiotic and antitumor activity and their randomization. FEMS Microbiol Rev. 2008, 32, 858–889. 37. Crespy, V.; Morand, C.; Besson, C.; Manach, C.; Démigné, C.; Rémésy, C. Comparison of the intestinal absorption of quercetin, phloretin and their glucosides in rats. J Nutr. 2001, 131, 2109–2114.

420

38. Schocker, N. S.; Portillo, S.; Brito, C. R.; Marques, A. F.; Almeida, I. C.; Michael, K. Synthesis of Galα (1, 3)

421

Galβ (1, 4) GlcNAcα-, Galβ (1, 4) GlcNAcα-and GlcNAc-containing neoglycoproteins and their

422

immunological evaluation in the context of Chagas disease. Glycobiology. 2015, 26, 39–50.



423 424 425 426 427 428 429 430

39. Pei, J.; Chen, A.; Zhao, L.; Cao, F.; Ding, G.; Xiao, W. One-pot synthesis of hyperoside by a three-enzyme cascade using a UDP-galactose regeneration system. J Agric Food Chem. 2017, 65, 6042-6048. 40. Huang, G. Synthesis and biological activities of galactose–aspirin conjugate prodrug designed for ADEPT and PMT. Med Chem Res. 2017 (https://doi.org/10.1007/s00044-017-2094-4) 41. Chen, C. C.; Guo, W. J. Isselbacher, K. J. Rat intestinal trehalase. Studies of the active site. Biochem. J. 1987, 247, 715-724. 42. Owens, D. K.; McIntosh, C. A. Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi. Phytochemistry. 2009, 70, 1382-1391.

431

43. Devaiah, S. P.; Owens, D. K.; Sibhatu, M. B.; Sarkar, T. R.; Strong, C. L.; Mallampalli, V. K.; Asiago, J.;

432

Cooke, J.; Kiser, S.; Lin, Z.; Wamucho, A. Identification, recombinant expression, and biochemical analysis of

433

putative secondary product glucosyltransferases from Citrus paradisi. J Agric Food Chem. 2016, 64, 1957-

434

1969.

435


Page 18 of 29

Page 19 of 29


436

Figure Legends

437

Scheme 1. Schematic diagram for one-pot multi-enzymes cofactors recycling (OPME-CR) galactosylation reaction

438

of diverse polyphenolic glycosides. Black box shows substrates transformed to galactose conjugated derivatives by

439

LgtB enzyme. Purple box shows cofactors recycling system. In cyan box-substrates and green box-products are

440

shown.

441

Figure 1. UHPLC-PDA and HR-QTOF ESI/MS spectra of one-pot multi-enzymes reactions of quercetin 3-O-β-D-

442

glucoside. A) UHPLC-PDA chromatograms of in vitro one-pot multienzymes reactions of quercetin 3-O-β-D-

443

glucoside to produce quercetin 3-O-β-D-lactoside at different time points: (i) 0 min, (ii) 30 min, and (iii) 60 min. B)

444

UV spectra of (i) quercetin 3-O-β-D-glucoside and (ii) the product. C) HR-QTOF ESI/MS analysis showing

445

production of quercetin 3-O-β-D-lactoside in positive ionization mode along with its mass fragmentation. D) The

446

possible structures of major fragments observed in positive ion mode.

447

Figure 2. Regular galactosylation reaction at different concentration of UDP-α-D-galactose and quercetin 3-O-β-D-

448

glucoside. 2mM of quercetin 3-O-β-D-glucoside was used with the increasing concentration of UDP-α-D-galactose.

449

Likewise 10mM of UDP-α-D-galactose was used for increasing concentration of quercetin 3-O-β-D-glucoside.

450

Figure 3. Optimized reaction conditions for OPME reaction for conversion of quercetin 3-O-β-D-glucoside to

451

quercetin 3-O-β-D-lactoside. A) Temperature, B) divalent metal ions, and C) pH.

452

Figure 4. Preparative scale OPME-CR galactosylation reaction with quercetin 3-O-β-D-glucoside for synthesis of

453

quercetin 3-O-β-D-lactoside. One millimolar quercetin 3-O-β-D-glucoside was added to the reaction mixture at 0.5,

454

1.5, and 2.5 h.

455

Figure 5. A) Structures of different polyphenolic glycosides modified in this study. B) Conversion percentage of

456

each substrate to respective D-galactose conjugated derivative at different time points.



Tables

Table 1. 1H-NMR of quercetin 3-O-β-D-glucoside, 14 and quercetin 3-O-β-D-lactoside

Position

Quercetin 3-O-β-D-glucoside 14

6 8 2′ 5′ 6′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′

6.20 (d, J=2.0 Hz, 1H) 6.39 ( d, J=2.0 Hz, 1H ) 7.70 ( d, J=2.0 Hz, 1H ) 6.86 ( d, J=8.5 Hz, 1H ) 7.58 ( dd, J=2.0, 8.5Hz, 1H ) 5.24 ( d, J= 8.0 Hz, 1H ) 3.40-3.70 (4H, m)

1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′

3.92 (1H, dd, J=12.0, 2.0 Hz) 3.56 (1H, dd, J=12.0, 5.0 Hz) -

Quercetin 3-O-β-Dlactoside 6.14 (s) 6.34 (s) 7.56 (m) 6.84 (d, J=8.26 Hz) 7.56 (m) 5.49(d, J=7.69 Hz) 3.0-4.0

4.21 (d, J=7.2Hz) 3.0-4.0


Page 20 of 29

Page 21 of 29


Table 2. 13C-NMR of quercetin 3-O-β-D-glucoside,31 and quercetin 3-O-β-D-lactoside Position 2 4 3 5 6 7 8 9 10 1′ 2′ 3′ 4′ 5′ 6′ 1′′ 2′′ 3′′ 4′′ 5′′ 6′′ 1′′′ 2′′′ 3′′′ 4′′′ 5′′′ 6′′′

Quercetin 3-Oβ-D-glucoside 156.58 177.9 133.77 161.70 99.12 164.61 93.97 156.63 104.43 122.06 115.67 148.92 145.27 116.66 121.63 101.31 74.55 76.96

Quercetin 3-Oβ-D-lactoside 156.94 177.51 133.56 161.58 99.64 166.39 94.26 156.41 103.72 121.41 115.70 149.20 145.37 116.47 121.99 101.22 74.28 76

70.39 78.03 61.43

80.94 75.77 60.86 104.29 73.68 75.22 68.61 70.97 60.86



Scheme 1.


Page 22 of 29

Page 23 of 29


Fig. 1 (A)

Fig. 1 (B)



Fig. 1 (C)

Fig. 1 (D)


Page 24 of 29

Page 25 of 29


Fig. 2



Fig. 3


Page 26 of 29

Page 27 of 29


Fig. 4



Fig. 5


Page 28 of 29

Page 29 of 29


Graphic for table of contents


One-pot Multi-enzyme Cofactors Recycling (OPME-CR) System for

Recommend Documents