bioconversion of pinoresinol into matairesinol using...

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Running title: PLR-SDH fusion protein expression in E. coli 1

Bioconversion of pinoresinol into matairesinol using recombinant 2

Escherichia coli 3

Han-Jung Kuo1, Zhi-Yu Wei1, Pei-Chun Lu1, Pung-Ling Huang2, and Kung-Ta Lee＊1 4

1Department of Biochemical Science and Technology, National Taiwan University, 5

2Department of Horticulture and Landscape Architecture, National Taiwan University 6

No. 1, Sec. 4, Roosevelt Road, Taipei, Taiwan. 7

*Corresponding author. 8

Tel.: +(886)-2-3366-4436 ; Fax: +(886)-2-2364-0961 9

E-mail address: [email protected] 10

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AEM Accepts, published online ahead of print on 21 February 2014Appl. Environ. Microbiol. doi:10.1128/AEM.03397-13Copyright © 2014, American Society for Microbiology. All Rights Reserved.

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Abstract 12

Lignans, a class of dimeric phenylpropanoid derivative found in plants, such as 13

whole-grain and sesame and flax seeds, have anticancer activity and can act as 14

phytoestrogens. The lignans secoisolariciresinol and matairesinol can, respectively, be 15

converted in the mammalian proximal colon into enterolactone and enterodiol, which 16

reduce the risk of breast and colon cancer. To establish an efficient bioconversion system 17

to generate matairesinol from pinoresinol, the genes pinoresinol reductase (PLR) and 18

secoisolariciresinol dehydrogenase (SDH) were cloned from Podophyllum pleianthum 19

Hance, an endangered herb in Taiwan, and the recombinant proteins, rPLR and rSDH, 20

were expressed in Escherichia coli and purified. The two genes plr-PpH and sdh-PpH 21

were also linked to form two bifunctional fusion genes, plr-sdh and sdh-plr, which were 22

also expressed in E. coli and purified. Bioconversion in vitro at 22°C for 60 minutes 23

showed that the conversion efficiency of fusion protein PLR-SDH was higher than that of 24

the mixture of rPLR and rSDH. The percentage conversion of (+)-pinoresinol to 25

matairesinol was 49.8% using PLR-SDH and only 17.7% using a mixture of rPLR and 26

rSDH. However, conversion of (+)-pinoresinol by fusion protein SDH-PLR stopped at 27

the intermediate product secoisolariciresinol. In vivo, (+)-pinoresinol was completely 28


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converted to matairesinol by living recombinant E. coli expressing PLR-SDH without 29

addition of cofactors. 30

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Introduction 32

Lignans, a large group of secondary metabolites found in plants, have a broad range of 33

medicinal functions. They have been identified in more than 60 families of vascular 34

plants (1) and are biosynthesized by the dimerization of two molecules of 35

phenylpropanoids. Different types of phenylpropanoid subunits, different coupling 36

orientations, and complicated modifications result in a wide diversity of lignan structures. 37

Lignans are also a major class of phytoestrogens with estrogen-like structures (2) and 38

antioxidant activity (3). Orally administered lignans have positive effects on breast cancer, 39

osteoporosis, and colon cancer (4). 40

The phytoestrogens pinoresinol, secoisolariciresinol, and matairesinol have been found 41

in whole-grain, vegetables, and fruits (5). Secoisolariciresinol and matairesinol are 42

converted into the enterolignans enterodiol and enterolactone by microflora in the 43

proximal colon in mammals and these two enterolignans are absorbed in the colon and 44

enter the body (6). The presence of enterolignans in serum can reduce the risk of breast, 45


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prostate, and colon cancer (7, 8). Enterolignans have weak estrogen-like activity and can 46

compete with estrogen for binding to estrogen receptors and inhibit the enzymes involved 47

in estrogen anabolism (9). High serum enterolignan levels can reduce the risks of acute 48

coronary syndrome (10). The concentrations of enterolignans in serum and urine are 49

indicators of plant lignan intake (11). 50

Podophyllotoxin, a type of aryltetralin lignan, is the precursor of the anti-cancer drugs 51

teniposide and etoposide (12-14). Pinoresinol-lariciresinol reductase (PLR), an 52

NADPH-dependent enzyme, is the key enzyme in podophyllotoxin biosynthesis (Fig. 1) 53

(15). The phenylpropanoid dimer (+)-pinoresinol is sequentially converted into 54

(+)-lariciresinol and (-)-secoisolariciresinol by PLR, then (-)-secoisolariciresinol is 55

converted into (-)-matairesinol by the NAD+-dependent secoisolariciresinol 56

dehydrogenase SDH (16), and this is, in turn, converted into podophyllotoxin by several 57

unidentified enzymes. 58

Podophyllum pleianthum, a herb traditionally used in Taiwan for treatment of snake 59

bite and wound healing, contains podophyllotoxin and, due to its slow growth and 60

overcollection, is now critically endangered (17). In this study, the genes plr and sdh were 61

cloned from P. pleianthum, fused, and heterologous expressed in E. coli to establish a 62


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high efficiency system for the conversion of (+)-pinoresinol to matairesinol. 63

Materials and Methods 64

Chemicals. The solvents and chemicals used were reagent or HPLC grade. 65

(+)-Pinoresinol was purchased from Arbonova (Turku, Finland), (+)-lariciresinol, 66

secoisolariciresinol, matairesinol, NADPH, NAD, DTT, and imidazole from 67

Sigma-Aldrich, and acetonitrile from Merck. TRIzol reagent and the RACE system were 68

from Invitrogen, the OneStep RT-PCR kit, pQE-30 Xa, and Ni-NTA Agarose from 69

Qiagen, and the restriction enzymes from New England Biolabs. 70

Cloning of plr-PpH and sdh-PpH. Aceptic P. pleianthum Hance was grown in B5 71

medium according to previous report (17), and the leaves were collected, frozen in liquid 72

nitrogen, and stored at -80°C. Total RNA was extracted using TRIzol reagent. The primer 73

sequences were listed in Table S1. Based on a multiple sequence alignment of PLR-Fi1 74

(GenBank accession number U81158) (15), PLR-Tp1 (AF242503) (18), PLR-Tp2 75

(AF242504) (18), PLR-La1 (AJ849358) (19), and PLR-Lu1 (AJ849359) (19), the 76

degenerate primers PLRFOR1, PLRFOR2, and PLRREV2 were designed to amplify 77

the conserved part of plr. RT-PCR was performed using a OneStep RT-PCR kit and 78

PLRFOR1 and oligo dT, and the PCR product further amplified using PLRFOR2 and 79


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PLRREV2. The resulting amplicon was sequenced and RACE-PCR performed to amplify 80

the cDNA from the conserved sequence to the 5’- or 3’-terminal. For 5’ RACE, the 81

primers PLR-GSP1-5’, PLR-GSP2-5’, and PLR-GSP3-5’ were designed and the 82

5’-terminal was synthesized using a 5' RACE System for Rapid Amplification of cDNA 83

Ends kit (Invitrogen), while PLR-GSP1-3’ was used for 3’ RACE PCR. The coding 84

region of plr-PpH was amplified using PLRF and PLRR. 85

The gene sdh-PpH was amplified using a OneStep RT-PCR kit and the forward primer 86

SDHF and reverse primer SDHR, designed based on GenBank accession number 87

AF352735 (16). 88

plr cDNA was amplified using primers sacPLRF and pstPLRR and introduced into 89

the expression vector pQE-30 Xa at the SacI and PstI restriction sites to generate 90

pQE-PLR. sdh-PpH cDNA was amplified using primers kpnSDHF and pstSDHR and 91

introduced into the pQE-30 Xa vector at the KpnI and PstI restriction sites to generate 92

pQE-SDH. pQE-PLR and pQE-SDH were transformed into E. coli M15 for heterologous 93

expression. 94

Generation of the plr-sdh and sdh-plr constructs. The proteins PLR-PpH and SDH-PpH 95

were linked using a (GGGGS)4 protein linker. SOEing PCR was performed to connect the 96


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cDNAs for plr-PpH, sdh-PpH, and the linker. Five primers, P-S1, P-S2, P-S3, P-S4, and 97

Link were designed to amplify plr-(linker)-sdh. P-S1 and P-S2 were used to amplify the 98

plr gene and P-S3 and P-S4 were used to amplify the sdh gene. The product of P-S3 and 99

P-S4 was further amplified using Link and P-S4 to obtain linker-sdh. The PCR products 100

containing plr and linker-sdh were mixed together and another 15 cycles of PCR reaction 101

performed without adding primers. The product was finally amplified with P-S1 and P-S4 102

to generate plr-(linker)-sdh. 103

sdh-(linker)-plr was also amplified using SOEing PCR. Five primers, S-P1, S-P2, S-P3, 104

S-P4, and Link were designed and S-P1 and S-P2 were used to amplify sdh, while S-P3 105

and S-P4 were used to amplify plr, which was further amplified using S-P4 and Link to 106

generate linker-plr, sdh and linker-plr were mixed and another 15-cycle PCR performed 107

without primer addition and the product amplified using S-P1 and S-P4 to generate 108

sdh-(linker)-plr. 109

plr-sdh and sdh-plr were introduced into the pQE-30 Xa vector at the SacI and PstI 110

restriction sites to generate pQE-PLR-SDH and pQE-SDH-PLR (Fig. S1) and E. coli 111

M15 was transformed with the vectors for heterologous protein expression. 112

Heterologous expression and protein purification. The recombinant E. coli M15 113


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strains were grown overnight at 37°C with shaking at 130 rpm in LB medium containing 114

100 ppm of ampicillin and 50 ppm of kanamycin (LBAK medium), then the bacteria 115

were inoculated into 100 mL of fresh LBAK medium in a Hinton flask and the cultures 116

grown under the same conditions to an OD600 of 0.6, when 0.01 mM IPTG was added to 117

induce heterologous protein expression. After 9 h of induction at 25°C with shaking at 118

130 rpm, the bacteria were harvested by centrifugation at 4,000× g for 30 min at room 119

temperature. The pellets from 100 mL of bacteria culture were resuspended in 10 mL of 120

lysis buffer (50 mM NaHPO4, 300 mM NaCl, 10 mM imidazole, pH 8.0) and the 121

suspensions sonicated in an ice bath to break the cells, then cell debris was removed by 122

centrifugation at 14,000× g for 30 min at 4°C. The target proteins in the supernatants 123

were purified using Ni-NTA agarose according to the manufacturer’s instructions and 124

stored at -20°C after addition of 50% glycerol (w/v). SDS-PAGE (12.5% polyacrylamide) 125

followed by Western blot with monoclonal Anti-polyHistidine antibody (Sigma) and goat 126

anti-mouse AP conjugate antibody was used to determine the molecular weight of the 127

recombinant proteins. 128

Enzyme assay. The enzyme activity assay for recombinant PLR (rPLR) was modified 129

from that in a previous report (20). The assay mixture (250 μL) containing 55 μM, 110 130


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μM, 167 μM, 223 μM and 279 μM (+)-pinoresinol or (+)-lariciresinol, 2.5 mM NADPH, 131

10 μg of rPLR, and 0.1 M potassium phosphate buffer, pH 7.1, was incubated at 30°C for 132

20 min. The kinetics of rPLR was determined by the disappearance of the substrates, 133

(+)-pinoresinol or (+)-lariciresinol. The enzyme activity assay for recombinant SDH 134

(rSDH) was modified from that in a previous report (16). The assay mixture (250 μL) 135

containing 0.4 μg of rSDH, 15 μM, 45 μM, 75 μM, 105 μM, 135 μM and 165 μM 136

(-)-secoisolariciresinol, 4 mM NAD, and 20 mM Tris buffer containing 5 mM DTT, pH 137

8.8, was incubated at 20°C for 4 min. The kinetics of rSDH was determined by the 138

formation of the product, matairesinol. Kinetic parameters were determined from 139

Lineweaver-Burk plots for both rPLR and rSDH. The assay conditions for the complete 140

transformation of (+)-pinoresinol to matairesinol were determined using rPLR and rSDH. 141

Assay mixtures containing 0.19 mM (+)-pinoresinol, 1 mM NADPH, 0.6 mM NAD, and 142

5 mM DTT in 20 mM Tris buffer at different pHs (7.0, 8.0 and 8.8) were tested at 143

temperatures of 22°C, 26°C, or 30°C for 1 h. The conditions chosen for assay of the 144

fusion proteins were 10 μg of PLR-SDH or SDH-PLR, 0.19 mM (+)-pinoresinol, 1 mM 145

NADPH, 0.6 mM NAD, and 20 mM Tris buffer containing 5 mM DTT, pH 8.0, at 22°C 146

for 1 h. To follow the time-course of conversion in vivo in bacteria, the assay mixture 147


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containing 2×109 or 1×1010 CFU of bacteria, 50 μM (+)-pinoresinol, and 20 mM Tris 148

buffer, pH 8.0, in LB medium was shaken vigorously at 22°C for 0.5, 1, 2, or 3 h. 149

HPLC analysis. The assay mixtures described above were extracted three times with 300 150

μL of ethyl acetate, air dried, and redissolved in 200 μL of methanol for HPLC analysis 151

using an Ascentis○R C18 HPLC column (particle size 5 μm, 25 cm x 4.6 mm) in an HPLC 152

system equipped with an SCL-10A system controller, SPD-M10A photodiode array 153

detector, SIL-10AD autosampler and LC-10AT pumps (all from SHIMADZU, Kyoto, 154

Japan). A binary buffer system (solvent A water containing 0.01% phosphoric acid, 155

solvent B acetonitrile) was used to create the mobile phase gradient. The analytes were 156

separated using 25% solvent B for the first 25 min, linear gradients of 25-43% solvent B 157

in 18 min, 43-55% in 3 min, 55-70% in 8 min, and 70-25% in 2 min, and 25% solvent B 158

for 4 min at a flow rate of 1 mL/min (20) with detection at 280 nm. 159

Results 160

Cloning and sequences analysis. A segment of plr in P. pleianthum Hance was amplified 161

by RT-PCR based on the conserved part of the plr sequences in Forsythia intermedia 162

(U81158), Thuja plicata (AF242503 and AF242503), Linum album (AJ849358), and 163

Linum usitatissimum (AJ849359), resulting in a 320-bp cDNA fragment, the deduced 164


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protein sequence of which showed high similarity to those of all the above PLRs and that 165

determined later for Linum perenne (20). A 933-bp cDNA containing the full length 166

coding sequence, plr-Pp (GeneBank Accession number KJ000045), was cloned by 5’- 167

and 3’-RACE PCR. The putative protein contained 311 amino acids. The deduced amino 168

acid sequence for PLR-PpH showed 75.2% identity and 85% similarity with the PLR of 169

Forsythia intermedi (Fig. S2). The SIM alignment tool was used to examine the sequence 170

for various motifs. A putative NADPH binding domain “GxxGxxG” was found in the 171

PLR-PpH sequence, as in the PLR of Forsythia intermedi. 172

A 834-bp cDNA containing the full length sdh-PpH coding sequence (GeneBank 173

Accession number KJ000044) was also identified based on the sdh sequence from 174

Forsythia intermedia (AF352735). The putative protein contained 278 amino acids. The 175

deduced amino acid sequence of SDH-PpH showed 98.2% identity and 99% similarity 176

with that in Podophyllum peltatum and contained a conserved NAD binding domain 177

“GxGGxG” (Fig. S3) . 178

Functional expression in E. coli. The gene plr-PpH was heterologously expressed in E. 179

coli M15 with an N-terminal 6-histidine tag. The recombinant PLR containing a 4 kDa 180

6-His-tag and Xa-factor had an apparent molecular weight of 39 kDa on PVDF 181


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membrane (Fig. 2A). Different concentrations of (+)-pinoresinol or (+)-lariciresinol were 182

added as substrate for an enzyme kinetic assay using 10 μg of rPLR and 2.5 mM NADPH 183

in 250 μL of assay mixture at 30°C, pH 7.1, and the Km and Vmax were found to be, 184

respectively, 19.8 μM and 7.34 μmol h-1 mg1 of protein for (+)-pinoresinol and 37.3 μM 185

and 3.12 μmol h-1 mg1 of protein for (+)-lariciresinol. 186

SDH-PpH was also heterologously expressed in E. coli M15 with an N-terminal 187

6-His-tag. The recombinant protein containing the His-tag and Xa-factor had an apparent 188

molecule weight of about 34 kDa on PVDF membrane (Fig. 2A). Different 189

concentrations of (-)-secoisolariciresinol were converted into matairesinol using 0.4 μg of 190

rSDH and 4 mM NAD in a 250 μL assay mixture at 20°C, pH 8.8, and the Km and Vmax 191

were found to be, respectively, 231 μM and 13.3 μmol min-1 mg1 of protein. The UV 192

absorption spectra for the products were shown to confirm the identities (Fig. S4). 193

Functional expression of the PLR-SDH fusion protein. Two fusion protein constructs 194

plr-sdh and sdh-plr were designed. The proteins were linked via a (GGGGS)4 protein 195

linker to maintain flexibility. The molecular weight of the fusion proteins PLR-SDH and 196

SDH-PLR including the N-terminal His-tag and Xa-factor was about 65 kDa on SDS gels 197

(Fig. 2A). 198


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To determine the best reaction conditions for rPLR and rSDH, different combinations 199

of buffers (20 mM Tris buffer at pH 7.0, 8.0, or 8.8) and temperatures (20ºC, 26ºC, and 200

30ºC) were tested using 30 μg of each of the recombinant proteins, 0.19 mM 201

(+)-pinoresinol, 1 mM NADPH, and 0.6 mM NAD. As shown in Fig. 2B, the results 202

show that a higher conversion was obtained at a lower reaction temperature and that the 203

efficiencies of formation of matairesinol and conversion of (+)-pinoresinol were both 204

highest in Tris buffer pH 8.0 at 22°C and these conditions were chosen for enzyme assay 205

of the fusion proteins. 206

As shown in Fig. 2C and Fig. 2D, (+)-pinoresinol was successfully converted into 207

matairesinol by PLR-SDH, but not by SDH-PLR. SDH activity was lost in SDH-PLR, 208

with the result that secoisolariciresinol, the product of PLR, accumulated in the reaction 209

mixture. Time-course conversion tests using the PLR-SDH or the mixture of rPLR and 210

rSDH are shown in Fig. 2E. At 60 min, the percentage conversion of (+)-pinoresinol to 211

matairesinol using PLR-SDH was 49.8%, higher than that of 17.7% using the rPLR and 212

rSDH mixture. 213

In vivo bioconversion by PLR-SDH. A further conversion assay was performed using 214

living recombinant E. coli. (+)-Pinoresinol was successfully converted to the final 215


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product matairesinol by the living bacteria expressing PLR-SDH in LB medium, pH 8.0, 216

with or without addition of cofactors NADPH and NAD at 22°C (Fig. 3). This result 217

suggested that the enzyme reactions could take place in the living cells without external 218

addition of cofactors. Cultures containing 2×109 or 1×1010 CFU of bacteria were used to 219

synthesize (-)-matairesinol in the absence of added cofactors. As shown in Fig. 4, the 220

substrate (+)-pinoresinol was almost completely (2×109 CFU) or completely (1×1010 221

CFU), converted into (-)-matairesinol by living cells without the addition of any cofactors 222

in 2 h. 223

Discussion 224

The enzyme PLR has been identified in Forsythia intermedia (15), Thuja plicata (18), 225

Linum perenne (20), L. album, and L. usitatissimum (19). We cloned plr cDNA from P. 226

pleianthum Hance. This is the first report of a PLR identified in the genera Podophyllum. 227

Podophyllum is the major plant source of podophyllotoxin. The plr-PpH sequence 228

showed high nucleotide sequence similarity with other plr sequences. Besides PLRs, 229

other lignan reductases, such as phenylcoumaran benzylic ether reductases (21) and 230

isoflavone reductases (22, 23), also show high sequence similarity with PLRs. All the 231

lignan reductases contain a conserved NADPH-binding domain “GxxGxxG” in a region 232


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close to the N-terminus and this NADPH-binding domain was also present in the deduced 233

amino sequence of PLR-PpH. Although pinoresinol stereospecificity was initially 234

reported to be determined by Leu164, Phe272, and Gly268 (24), these residues were soon 235

found not to be sufficient (19). Thus, the existence of Gly268 and another aromatic amino 236

acid, Tyr272, instead of Phe in the PLR-PpH sequence is not sufficient to explain the 237

(+)-pinoresinol substrate stereospecificity and suggests that other residues are also 238

involved. The Km value of rPLR for pinoresinol was 37.3 μM which was close to that of 239

PLR-Fi1 and PLR-Fi2 (27 and 23 μM respectively) (15). However, the Km for 240

lariciresinol was 37.3 μM which was lower than that of PLR-Fi1 and PLR-Fi2 (121 and 241

123 μM respectively) (15). It seems the affinity of rPLR to lariciresinol was slightly 242

higher than the PLRs in F. intermedia. 243

The enzyme SDH has been identified in F. intermedia and P. peltatum (16) and its 244

cDNA (sdh-PpH) was cloned from P. pleianthum Hance in the present study. The 245

deduced amino acid sequence showed high similarity with other SDH sequences, and the 246

conserved NAD-binding domain “GxGGxG” and the amino acid residues Ser153, Tyr167, 247

and Lys171, predicted to be involved in the SDH catalytic center (25) were found to be 248

present. The Km value of rSDH was 231 μM which was higher than that of SDH-Pp (160 249


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μM) (16). This may suggest that the substrate affinity of rSDH was lower than the SDH 250

in P. peltatum. The recombinant protein was able to convert (-)-secoisolariciresinol into 251

matairesinol. Since no PLR and SDH active on the opposite stereoisomers were found, 252

our results suggest that (+)-pinoresinol, (+)-lariciresinol, (-)-secoisolariciresinol, and 253

(-)-matairesinol are involved in the podophyllotoxin biosynthesis pathway in P. 254

pleianthum Hance. 255

In order to simplify the bioconversion process from (+)-pinoresinol to matairesinol, 256

fusion proteins were designed. The enzyme reaction conditions used for the fusion 257

proteins were a pH of 8.0 (between the values used for rPLR and rSDH individually) and 258

a relatively low reaction temperature of 22°C. This low temperature is not surprising, 259

since P. pleianthum Hance grows in medium to high altitude mountainous areas. PLR 260

activity was seen with both the PLR-SDH and SDH-PLR fusion proteins, whereas SDH 261

activity was seen only with PLR-SDH. This suggests that the C-terminal region of SDH 262

is important for its enzyme function. A previous report of the crystal structure of SDH 263

also suggested that substrate-enzyme binding requires the involvement of a C-terminal 264

flexible arm (25). Thus, a decreased flexibility in the C-terminus of SDH might be the 265


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reason for its disrupted function in SDH-PLR and this could possibly be overcome if a 266

longer and more flexible protein linker was used. 267

The percentage conversion of (+)-pinoresinol to matairesinol catalyzed by PLR-SDH 268

was higher than that using a mixture of rPLR and rSDH. The distance from PLR to SDH 269

was restricted in the fusion protein by the flexible protein linker (GGGGS)4. This may 270

make it easier for (-)-secoisolariciresinol, the product of PLR and the substrate for SDH, 271

to move from PLR to SDH, suggesting that a metabolic channel, in the broad sense, was 272

formed in the fusion protein to increase conversion efficiency, as suggested previously 273

(26-31). The same phenomenon of an increase in conversion efficiency in fusion proteins 274

has been reported for isoflavone synthase-chalcone isomerase (32) and 275

trehalose-6-phosphate synthetase-trehalose-6-phosphate phosphatase (33) fusion proteins. 276

We also found that the substrate (+)-pinoresinol was converted into matairesinol in 277

medium containing living E. coli transformants expressing PLR-SDH without addition of 278

cofactors, such as NAD and NADPH, suggesting that the recombinant enzymes utilize 279

the cofactors produced by the living cells. This might result from the substrate entering 280

the living cell or broken cells releasing enzyme and cofactors into the culture medium. To 281

distinguish between these two processes, further experiments are needed. In conclusion, 282


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we have established an E. coli bioconversion system for the efficient conversion of plant 283

lignan (+)-pinoresinol to matairesinol without the addition of cofactors to save time and 284

cost. 285

Acknowledgements 286

This work was supported by grant 96-2313-B-002-070-MY3 from the National 287

Science Council, Executive Yuan, Taiwan, ROC. 288

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Figure legends 382

Fig. 1 Biosynthesis pathway of podophyllotoxin. 383

Fig. 2 Expression and functional analysis of rPLR, rSDH and the fusion proteins. (A) 384

Western blot detection of the recombinant proteins with anti-His antibody. Lane 1 is 385

rPLR (~39 kDa), lane 2 is rSDH (~34 kDa), lane 3 is PLR-SDH fusion protein (~65 kDa) 386

and lane 4 is SDH-PLR fusion protein (~65 kDa). NC is the protein extract from wild 387

type E. coli M15 as the negative control. (B) Bioconversion efficiency under different 388

conditions using rPLR and rSDH (1 h reaction). The concentration of (+)-pinoresinol is 389

indicated by white bars, that of lariciresinol by light gray bars, that of secoisolariciresinol 390

by dark gray bars, and that of matairesinol by black bars. The assay mixture without 391

protein was used as the negative control. (C)-(D) HPLC traces at 280 nm of lignan 392

compounds extracted from the 1 h bioconversion assay mixture using the two different 393

fusion proteins. The substrate (+)-pinoresinol was converted to the final product 394

matairesinol using fusion protein PLR-SDH (C), but not using SDH-PLR (D). (E) 395

Percentage bioconversion of matairesinol using a mixture of rPLR and rSDH or 396

PLR-SDH fusion protein. The results using PLR-SDH are shown by dark gray bars and 397

those using the rPLR and rSDH mixture by light gray bars. The error bars represent 398


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standard deviation. 399

Fig. 3 HPLC traces at 280 nm of lignan compounds extracted from conversion assay 400

using living recombinant E. coli. The substrate (+)-pinoresinol was co-incubated for 6 h 401

with wild type E. coli (A), or with E. coli expressing PLR-SDH in the existence (B) or 402

absence (C) of cofactors. 403

Fig. 4 Time course of in vivo bioconversion of (+)-pinoresionol to matairesinol by E. coli 404

M15 transformed with pQE-PLR-SDH. (A) Percentage of substrate (+)-pinoresionol 405

remaining. (B) Production of the final product matairesinol. The enzyme assay was 406

performed at two different concentrations of bacteria, as indicated by the dark gray and 407

light gray bars. The error bars represent standard deviation. 408

409


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