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

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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

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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]

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ABSTRACT

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

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

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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

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

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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

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

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

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efficiently and cost-effectively.

12 13

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

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INTRODUCTION

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

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

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

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

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

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

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

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

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

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

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

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

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

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infections.9,10

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

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

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

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

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

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

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galactose

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

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

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

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

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

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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

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Previously,

the

β-1,4-

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

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carrying lactose moiety has not been reported yet.

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

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

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

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

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

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

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

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

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

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

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MATERIALS AND METHODS

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Culture media and chemicals

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

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

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

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

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

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

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

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

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Recombinant enzymes expression and purification

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Regular galactosylation using LgtB enzyme

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

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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-

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

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

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QTOF-ESI/MS analyses.

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

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

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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

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

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

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the different substrate concentrations.

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

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

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

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

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

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

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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-

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

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

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

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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

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

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UHPLC-PDA.

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

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

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

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

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

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

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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

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

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

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

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

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

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

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of UMP.

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OPME-CR galactosylation reaction with other polyphenolic glycosides

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

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

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

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

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

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

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and LgtB) at similar concentrations as described above.

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

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

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

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

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Analytical methods

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

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

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

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

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

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

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

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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

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

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

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

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

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

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substrate and product in HPLC chromatogram.

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

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

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

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

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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

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were

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

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

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software.

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

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

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

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

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

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

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

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

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

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RESULTS

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Expression and preparation of enzymes

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

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

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

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

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for the reactions.

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

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

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

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

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

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

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

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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

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300°C;

desolvation

gas

flow,

C-NMR) and two-dimensional NMR

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

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

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Structural analysis

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

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

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

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

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

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

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

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

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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

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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

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

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

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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-

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

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

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

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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

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

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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,

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

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

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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

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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

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

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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

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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

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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-

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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

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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.

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Figure Legends

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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.

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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

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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

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