1

Supplementary Data of 1

2

Biosynthesis of novel phloretin glucosides 3

4

Ramesh Prasad Pandey1, Tai Feng Li1, Eun-Hee Kim2, Tokutaro Yamaguchi1, Yong 5

Il Park3, Joong Su Kim4, Jae Kyung Sohng1* 6

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1Institute of Biomolecule Reconstruction (iBR), Department of Pharmaceutical Engineering, 8

Sun Moon University, 100, Kalsan-ri, Tangjeong-myeon, Asansi, Chungnam 336-708, Korea 9

2Division of Magnetic Resonance, Korea Basic Science Institute, Ochang, Chungbuk 363-883, 10

Korea 11

3Department of Biotechnology, The Catholic University of Korea, Bucheon, Gyeonggi-do 12

420-743, Korea 13

4Bioindustry Process Center, Jeonbuk Branch Institute, Korea Research Institute of 14

Bioscience and Biotechnology, Jeonbuk 580-185, Jeong-Ub, Korea 15

16 17

2

Supplementary Materials and Methods 18

Bacterial strains, vectors, media 19

Bacillus licheniformis ATCC14580 was used for the isolation of genomic DNA. E. coli XL1 20

Blue MRF’ (Stratagene, USA) was used for DNA manipulation, and E. coli BL21 (DE3) host 21

(Stratagene) was used for the expression of protein. All E. coli strains were grown at 37°C in 22

Luria Bertani (LB) liquid or plate supplemented with ampicillin antibiotic (100 µg mL−1) 23

when required. pGEM®-T Easy vector (Promega, USA) was used for the cloning of PCR 24

products and pET28a (+) vector was used as an expression vector. All DNA manipulation was 25

performed by using standard techniques for Escherichia coli (1). The restriction enzymes 26

were from Takara (Shiga, Japan), while phloretin and UDP-glucose used in this study were 27

purchased from Sigma. 28

Cloning and expression of yjiC 29

The yjiC gene was amplified by using primer pairs: yjiC-F/yjiC-R (Supplementary table S1). 30

The 1,191 bp long fragment of yjiC (sequence correct) was excised from pGEM®-T vector 31

by BamHI/XhoI and was cloned into the same sites of pET28a (+) to generate the 32

recombinant expression vector pET28-YjiC. pET28-YjiC was transformed into E. coli BL21 33

(DE3) using the heat shock transformation method. 50 µL of E. coli BL21 (DE3) harboring 34

pET28-YjiC was cultured overnight and was inoculated into 50 mL LB medium and 35

incubated at 37°C until absorbance at 600 nm (OD600) reached to 0.5-0.7. The culture was 36

induced with 0.5 mM of isopropyl-β-D-thiogalactopyranoside (IPTG) followed by continued 37

growth at 20°C with shaking for 20 h. The cells were harvested by centrifugation at 3,000 38

rpm for 10 min, washed twice with 100 mM Tris-Cl (pH: 8.0) and resuspended in 1 mL of the 39

same buffer. The cells were lysed by sonication and the lysate was cleared by centrifugation 40

(12,000×g) for 30 min at 4°C. Cleared lysate was mixed with 1 mL of Ni-nitrilotriacetic acid 41

3

(Ni-NTA) agarose (Qiagen) and was purified according to the manufacturer’s instructions. 42

Fractions containing purified protein were assessed via sodium dodecyl sulfate-43

polyacrylamide gel electrophoresis (12% SDS-PAGE), and were concentrated using Amicon 44

Ultra-15 (Millipore, 10K NMWL centrifugal filters). The purified protein was stored in the 45

buffer containing 100 mM Tris-HCl (pH 8.0) and 20% [v/v] glycerol until use. Protein 46

concentration was determined by the Bradford assay. 47

Glycosyltransferase activity assay 48

The reactions for YjiC were carried out in a total volume of 100 µl of reaction mixture 49

containing100 mM Tris-Cl (pH 8.8), 2 mM UDP-glucose, 2 mM phloretin, 2 mM MgCl2 and 50

40 µg/mL of appropriately diluted enzyme. The reaction mixture was incubated at 30°C for 4 51

h. The assay mixtures lacking enzyme served as controls. The reaction was quenched by 52

heating the reaction mixtures to 100°C for 1 min, centrifuged to remove proteins, and the 53

reaction product was monitored by HPLC-PDA after proper dilution. 54

Whole-cell biocatalysis 55

The engineered host E. coli BL21 (DE3)/ΔpgiΔzwfΔushA harboring recombinant plasmid 56

was used for the whole cell biocatalyst assay. 200 µL of overnight culture of the above strain 57

was transferred to 50 mL of LB liquid medium (with antibiotics for triple mutants, 58

supplemented with 1% mannitol) and was incubated at 37⁰C until the optical density at 600 59

nm (OD600nm) reached approximately 0.6. Then, 0.5 mM of IPTG was added to the final 60

concentration, and the cultures were incubated at 25°C for 6 h. The cells were aseptically 61

centrifuged at room temperature, and the cell pellets were resuspended in terrific broth (TB) 62

medium (15 mL). 500 µL of each cell suspension was transferred to wells of a 1 mL 96 well 63

plate. To the same cultures 100 µM, 250 µM, and 500 µM of phloretin (final concentration) 64

was fed, and they were then incubated at 25°C/500 rpm in a Thermoshaker. Samples were 65

4

taken at different time intervals, were centrifuged, and were extracted by double volume of 66

ethyl acetate. The organic layer was dried and was again dissolved in 500 µL of HPLC grade 67

methanol for HPLC-PDA analysis. 68

Product analysis and elucidation 69

Reverse-phase HPLC-PDA analysis was performed with a C18 column (YMC-Pack ODS-70

AQ (4.6x150, 5μm) connected to a photo diode array (288 nm) using an isocratic condition of 71

70% H2O (0.1% trifluroacetic acid buffer) and 30% acetonitrile (ACN) at a flow rate of 2 72

mL/min. The molecular weight of the products were confirmed by LC-QTOF-ESI-MS/MS 73

[ACQUITY (UPLC, Waters Corp., USA)-SYNAPT G2-S (Waters Corp., USA)]. The 74

products were purified by prep-HPLC with a C18 column (YMC-Pack ODS-AQ (150 x 20 75

mm I. D., 10 μm) connected to a UV detector (288 nm) using a 36 min binary program with 76

ACN 20% (0-5 min), 40% (5-10 min), 40% (10-15 min), 90% (15-25 min), 90% (25-30 min), 77

and (30-35 min) 10% at a flow rate of 10 mL/min. The purified products were then dried 78

completely in a lyophilizer and were used for structural elucidation. Nuclear magnetic 79

resonance (NMR) spectra were obtained in methanol-d4 (Aldrich, USA) using 800 and 500 80

MHz Bruker, BioSpin when appropriate. 81

82

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Supplementary Table Legends 83

Table S1. Primers used in this study. 84

Table S2.Strains and vectors used and constructed in this study. 85

Table S3. Reaction conditions for in vitro reaction at different donor substrate concentrations 86

and time of incubation. Reaction incubation time 4h for reactions 1 to 4 and 12 h for reactions 87

5 to 8. All the reactions were incubated at 30oCand stopped by heating at 100oC for 1 min. 88

89

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Table S1. 90

Primers Oligonucleotide Sequences (5’-3’) Restriction sites

yjiCF

yjiCR

GGATCCATGGGACATAAACATATCGCG

CTCGAGTTATTTTACTCCTGCGGGTGCTAA

BamHI

XhoI

91

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Table S2. 92

Strains/Plasmids Description Source/reference Strains Escherichia coli XL-1 Blue (MRF’) General cloning host Stratagene, USA BL21(DE3)

BL21(DE3)/ΔpgiΔzwfΔushA BL21 (DE3)/ pET28-YjiC B L 2 1 ( D E 3 ) / Δpgi Δzwf

ΔushA/ pET28-YjiC

ompT hsdT hsdS (rB- mB

-) gal (DE3) BL21(DE3)/pgi, zwf, and ushAdeleted BL21(DE3) carrying pET28-YjiC, yjiCglycosyltransferaseover-expression BL21(DE3)/ ΔpgiΔzwfΔushA carrying pET28-YjiC, yjiC glycosyltransferase over-expression

Novagen [20] This study This study

Plasmid vectors pGEM®-T easy vector pET28a (+)

General cloning vector,T7 and SP6 promoters, f1 ori, Ampr Expression vector,, T7 promoter, pBR322 ori, Ampr

Promega, USA Novagen

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94

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Table S3. 95

Total reaction volume: 100μl Tris-HCI buffer MgCl2 UDP-Glucose Phloretin Protien (YjiC)

Phloretinin vitro reaction 1 50 mM 2mM 2mM 2mM 40μg/mL Phloretinin vitro reaction 2 50 mM 2mM 4mM 2mM 40μg/mL Phloretinin vitro reaction 3 50 mM 2mM 8mM 2mM 40μg/mL Phloretinin vitro reaction 4 50 mM 2mM 16mM 2mM 40μg/mL Phloretinin vitro reaction 5 50 mM 2mM 2mM 2mM 40μg/mL Phloretinin vitro reaction 6 50 mM 2mM 4mM 2mM 40μg/mL Phloretinin vitro reaction 7 50 mM 2mM 8mM 2mM 40μg/mL Phloretinin vitro reaction 8 50 mM 2mM 16mM 2mM 40μg/mL

96

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Supplementary Figure Legends 97

Figure S1. Amino acid sequences alignment of YjiC with other different bacterial 98

glycosyltransferases.The BLAST analysis and sequence alignment study with previously 99

known bacterial glycosyltransferases has different level of amino acid identities and 100

similarities. YjiC has 31%, 33%, and 38% identities with BcGt-3 from Bacillus cereus ATCC 101

14580 (GenBank: AAS41737.1), OleD from Streptomyces antibioticus (UniportKB accession: 102

Q53685), and CalG4 from Micromonospora echinospora (GenBank: AAM70365.1) 103

respectively. The similarities of YjiC were 53%, 52% and 56% respectively with BcGt-3, 104

OleD, and CalG4 respectively. 105

Figure S2. pET28a-YjiC recombinant expression vector harboring yjiC glycosyltransferase 106

gene. The gene yjiC was cloned in BamHI/XhoI site of the expression vector pET28a (+). 107

Figure S3. Expression and purification of the recombinant YjiC protein by Ni-NTA resin. 108

Lane 1: Insoluble protein fraction; Lane 2:Soluble protein fraction; Lane 3:Protein marker; 109

Lane 4: Eluted protein by 100mM imidazole; and Lane 6: Eluted protein by 200mM 110

imidazole. 111

Figure S4. Comparative HPLC chromatogram for the in vitro reactions 1 to 4 (Table S3). 112

Area percentage of each peak is given in the table below the chromatogram. 113

Figure S5.Comparative HPLC chromatogram for the in vitro reactions 5 to 8 (Table S3). 114

Area percentage of each peak is given in the table below the chromatogram. In reaction 8, a 115

novel peak P4 is seen in between P3 and P5. 116

Figure S6. Comparative study of bioconversion of phloretin and production optimization at 117

different substrate concentrations at different time interval with E. coli BL21 118

(DE3)/ΔpgiΔzwfΔushA/pET28-YjiC) Production of different products when 100 µM of 119

phloretin was supplemented. B) Production of different products when 250 µM of phloretin 120

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was supplemented. 121

Figure S7. Structural elucidation of product P1 as Phloretin-4’-O-glucoside. a) 1H NMR, b) 122

13C NMR, c) 1H-1H COSY, d) HSQC, e) HMBC, and f) ROSEY. 123

Figure S8. Structural elucidation of product P2 as Phloretin-2’-O-glucoside. a) 1H NMR, b) 124

13C NMR, c) 1H-1H COSY, d) HSQC, e) HMBC, and f) ROSEY. 125

Figure S9. Comparing 1H-NMR of all the five phloretin glucosides products (P1, P2, P3, P4, 126

and P5) from buttom to top respectively. P3 and P4 were identified in mixture. 127

Figure S10. 1H-NMR analysis of P3 (Phloretin 4’,4-O-diglucoside) and P4 (Phloretin 6’,4-O-128

diglucoside). 129

Figure S11. Structural elucidation of compound P5 (Phloretin2’, 4’, 4-O-triglucoside). a) 1H-130

NMR analysis. b) Comparing 1H-NMR of all the three novel phloretin glucosides products 131

(P3, P4, and P5). P3 and P4 were identified in mixture. c) Comparision of aglycone part and d) 132

comparision of glucose part. 133

134

135

136

137

Figure

S1.

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138

139

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Figure

S2.

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141

142

143

Figure

S3.

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144

145146

Figure

S4.

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147

148

149

Figure

S5.

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150

151

152

153

Figure

S6.

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Structural elucidation of phloretin glucosides products 154

Figure S7 155

a) 156

157

1H NMR (800 MHz, Methanol-d4) δ 6.95 – 6.91 (m, 2H), 6.59 (d, J = 8.4 Hz, 2H), 5.99 (d, J 158

= 1.7 Hz, 2H), 4.83 (dd, J = 7.6, 1.6 Hz, 1H), 3.81 (dd, J = 12.2, 2.3 Hz, 1H), 3.62 (dd, J = 159

12.2, 5.5 Hz, 1H), 3.37 (t, J = 9.3 Hz, 1H), 3.36 (t, J = 9.3 Hz, 1H), 3.35 (dd, J = 9.3, 7.5 Hz, 160

1H), 3.30 (t, J = 9.2 Hz, 1H), 3.19 (dd, J = 8.8, 7.0 Hz, 2H), 2.75 (t, J = 7.8 Hz, 2H). 161

162

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b) 163

164

13C NMR (201 MHz, MeOD) δ 207.13, 165.06, 164.85, 156.51, 133.96, 130.43, 116.23, 165

106.99, 101.21, 96.55, 78.34, 77.99, 74.73, 71.25, 62.49, 47.61, 31.31. 166

167

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c) 168

169

170

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d) 171

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173

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e) 174

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176

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f) 177

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179

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Figure S8 180

a) 181

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1H NMR (800 MHz, Methanol-d4) δ 7.10 – 7.02 (m, 2H), 6.74 – 6.64 (m, 2H), 6.18 (d, J = 183

2.0 Hz, 1H), 5.95 (d, J = 2.2 Hz, 1H), 5.04 (d, J = 7.4 Hz, 1H), 3.91 (dd, J = 12.2, 2.3 Hz, 184

1H), 3.72 (dd, J = 12.2, 5.6 Hz, 1H), 3.47 (dd, J = 9.3, 7.4 Hz, 1H), 3.45 (t, J = 9.3 Hz, 1H), 185

3.49 – 3.42 (m, 2H), 3.47 – 3.43 (m, 1H), 3.38 (t, J = 9.1 Hz, 1H), 2.87 (dtd, J = 11.5, 7.1, 6.7, 186

4.4 Hz, 2H). 187

188

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b) 189

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13C NMR (201 MHz, Methanol-d4) δ 206.67 , 167.71 , 166.16 , 162.45 , 156.53 , 134.02 , 191

130.52 , 116.22 , 106.91 , 102.23 , 98.50 , 95.61 , 78.65 , 78.58 , 74.86 , 71.25 , 62.58 , 47.11 , 192

31.00 . 193

194

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c) 195

196

197

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d) 198

199

200

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e) 201

202

203

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f) 204

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206

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Figure S9 207

208

209

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Structural elucidation of compound P3and P4 210

Figure S10. 211

212

1H-NMR (500 MHz, MeOD): δ 7.16 (dd, J = 12.2,8.3 Hz, 4H), 7.01 (dd, J = 8.6, 2.9 Hz, 4H), 213

6.19 (d, J= 2.0 Hz, 1H), 6.09 (s, 1H), 5.96 (d, J = 2.2 Hz, 1H), 4.98 (dd, J = 54.9, 6.7 Hz, 214

2H), 4.89-4.79 (m, 2H). Other signals in sugar regions are fairly complex because of mixture 215

of two diglucosides which are as follows 4.01-3.80 (m, 4H), 3.70 (ddd, J =11.9,9.2,5.2 Hz, 216

4H), 3.55-3.21 (m, 24 H), 2.92 (q, J=7.8 Hz, 4H). Since the two products (P3 and P4) are 217

diglucosides, their separation by preparative HPLC was quite difficult. Hence we 218

characterized those products by NMR analysis. We apologize to report both compounds in a 219

single chromatogram. 220

221

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Figure S11. Structural elucidation of compound P5 222

a) 223

224

1H-NMR (500 MHz, MeOD): δ 7.27-7.10 (m, 2H), 7.06-6.91 (m, 2H), 6.47 (d, J = 2.4 Hz, 225

1H), 6.24 (d, J=2.3 Hz, 1H), 5.17-5.07 (m, 1H), 5.01 (d, J = 7.1 Hz, 1H), 4.92-4.79 (m, 1H), 226

Glucose region: δ (4.01- 2.9). 227

228

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b) 229

230

231

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c) 232

233

234

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d) 235

236

237

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References 238

1. Sambrook, J., E. F. Fritsch, T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual, 239

Vol.5 2nd ed., Cold Spring Harbor Laboratory Press, NewYork. 23 240

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