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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
11
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
31
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
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