Appl Microbiol Biotechnol (2014) 98:8165–8177 DOI 10.1007/s00253-014-5934-x
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana Masayuki Tamura & Yukiko Tsuji & Tatsuya Kusunose & Atsushi Okazawa & Naofumi Kamimura & Tetsuya Mori & Ryo Nakabayashi & Shojiro Hishiyama & Yuki Fukuhara & Hirofumi Hara & Kanna Sato-Izawa & Toshiya Muranaka & Kazuki Saito & Yoshihiro Katayama & Masao Fukuda & Eiji Masai & Shinya Kajita
Received: 8 April 2014 / Revised: 2 July 2014 / Accepted: 5 July 2014 / Published online: 24 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type
and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected
Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5934-x) contains supplementary material, which is available to authorized users. M. Tamura : Y. Tsuji : K. Sato-Izawa : S. Kajita (*) Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan e-mail: [email protected] T. Kusunose : A. Okazawa : T. Muranaka Graduate School of Engineering, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan N. Kamimura : Y. Fukuhara : M. Fukuda : E. Masai Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan T. Mori : R. Nakabayashi : K. Saito Metabolomics Research Group, RIKEN Center for Sustainable Resource Science, RIKEN, 1-7-22, Tsurumi, Kanagawa 230-0045, Japan S. Hishiyama Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan
H. Hara Department of Environmental Engineering and Green Technology, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Kuala Lumpur 54100, Malaysia
K. Saito Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
Y. Katayama College of Bioresource Sciences, Nihon University, 1866 Mameino, Fujisawa, Kanagawa 252-0880, Japan Present Address: A. Okazawa Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, NakakuSakai Osaka 5998531, Japan
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levels of lariciresinol in the transgenic plant following βglucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.
et al. 2013; Suzuki and Umezawa 2007). Most lignans are biosynthesized enantiospecifically. For example, in Forsythia intermedia, two molecules of coniferyl alcohol are dehydrogenated by an oxidase such as laccase to give two coniferyl alcohol radicals, and then, they are coupled under the control of the dirigent protein, which plays the important role of structural regulator, and (+)-Pin is preferentially generated (Davin et al. 1997). (+)-Pin is further converted to (−)secoisolariciresinol (Sec) through (+)-lariciresinol (Lar) in a reaction catalyzed by pinoresinol/lariciresinol reductase (PLR; Dinkova-Kostova et al. 1996). In Arabidopsis thaliana, pinoresinol reductase 1 (AtPrR1) can reduce both (+)- and (−)Pin to generate (+)- and (−)-Lar (Nakatsubo et al. 2008). Lignans exhibit a broad range of biological activities, including anti-viral, anti-fungal, anti-bacterial, and antioxidant activity, root elongation inhibition, and anti-feedant activity against insects (Deyama and Nishibe 2010). Although these activities suggest that lignans are important for plant protection against biotic and abiotic stresses, most are still under investigation (Harmatha and Dinan 2003; Lorenc-Kukuła et al. 2009). Lignans are also characterized as compounds with a variety of pharmaceutical applications. For example, the seeds of flax contain high levels of secoisolariciresinol diglucoside (SDG), which displays a wide range of health-
Keywords Arabidopsis thaliana . Lignan . Pinoresinol reductase . Sphingobium sp. SYK-6
Introduction Lignans are common secondary metabolites in plants. It is synthesized through the coupling of phenylpropanoid monomers such as coniferyl alcohol, linked through the 8 and 8′ carbons in side chains of the monomers. A large variety of lignans with different chemical structures have been isolated from plants, and they are categorized based on their structure such as furan, furofuran, and dibenzylbutane. Among the wide range of lignans, molecules synthesized through pinoresinol (Pin) have been structurally characterized, and their biosynthetic pathways have been extensively investigated (Fig. 1; Davin and Lewis 2003; Davin et al. 2008; Fuss 2003; Satake
Neolignans 7′
6′ 9 7
Derivatives
1′
5′
8′
8
9′
Lignans
2′
4′
3′
1 6
2
5
3
f eruloyl ether
4
?
Aromatic Amino Acid
Lariciresinol monoglucoside (Lar-Glc)
Dehydrodiconiferyl alcohol
?
3 2
4
9 1 8 7
PinZ AtPrR1
8 8′
2 3
5
PinZ
AtPrR1, AtPrR2
6
9′ 1
6
5
7
9
6′ 1′
5′
4 4′
2′ 3′
7′
Lariciresinol (Lar)
Pinoresinol (Pin)
Secoisolariciresinol (Sec)
Conif eryl alcohol
Ferulic acid
?
Feruloyl-CoA
Conif eraldehyde
Epipinoresinol (Epi)
Syringaresinol gucosides
PinZ Syringaresinol Sinapic acid
Secoisolariciresinol diglucosides (SDG)
Matairesinol (Mat)
Dimethoxylariciresinol glucosides Dimethoxylariciresinol
Sinapyl alcohol
Fig. 1 The predicted biosynthetic pathway of lignans, neolignans, and related compounds in Arabidopsis thaliana and catalytic steps catalyzed by PinZ. AtPrR1 and AtPrR2 are endogenous pinoresinol reductases of A. thaliana
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promoting activities including radical-scavenging and anticancer effects (Lainé et al. 2009; Prasad 1997). In addition, podophyllotoxin, isolated from Podophyllum plants, is a precursor used to produce drugs targeting testicular cancer and small cell lung carcinoma (Damayanthi and Lown 1998). Lignans, including Pin, Lar, and Sec, are thought to be metabolized by mammalian intestinal bacteria to produce enterodiol and enterolactone, which modulate immune responses and have been classified as mammalian lignans (Heinonen et al. 2001; Kurzer and Xu 1997; Landete 2012; Parr and Bolwell 2000). Structural transformations of lignan compounds by intestinal bacteria have been characterized, and some enzymatic functions are known. For example, Enterococcus faecalis strain PDG-1 can convert (+)-Pin to (+)-Lar, and both Pin and Lar are transformed to Sec by Eggerthella lenta (Clavel et al. 2006; Xie et al. 2003). These observations suggest that intestinal bacteria possess identical types of catabolic enzymes for lignan transformation as do plants. However, few catabolic genes for these lignan transformations had been cloned from bacteria, including intestinal bacteria. Recently, we successfully cloned two genes, pinZ and Saro_2808, for pinoresinol reductase (PrR) from the bacteria Sphingobium sp. SYK-6 and Novosphingobium aromaticivorans DSM 1244, respectively (Fukuhara et al. 2013). This was the first report of the cloning of bacterial PrR genes. Although the deduced amino acid sequence of pinZ shares no significant identity with that of plant-origin PrR or PLR, recombinant PinZ catalyzed the reduction of (±)Pin to (±)-Lar and of (±)-syringaresinol to (±)dimethoxylariciresinol in the presence of NADPH (Fukuhara et al. 2013). In addition, the specific activity of recombinant PinZ toward (±)-Pin was over 1,000-fold higher than that of recombinant AtPrR1, reported by Nakatsubo et al. (2008). PinZ also has Lar-reducing activity, although this activity is weaker than a similar activity toward Pin (Fukuhara et al. 2013). In the present study, pinZ was overexpressed in transgenic A. thaliana plants under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter with the aim of modulating lignan accumulation. In previous studies, metabolic engineering of lignan accumulation has already been achieved in some transgenic plants. Ayella et al. (2007) and Banerjee and Chattopadhyay (2010) reported the overaccumulation of SDG and phyllanthin in transgenic wheat and Phyllanthus amarus plants, respectively, overexpressing PLR genes of plant origin. Furthermore, sesamin accumulation has been achieved by a combination of the downregulation of PLR and the overexpression of Sesamum CYP81Q1 genes in transgenic Forsythia cell cultures (Kim et al. 2009). Accumulation of hinokinin, which is synthesized via matairesinol (Mat), was almost completely suppressed in hairy roots of Linum corymbulosum with the RNA interference construct for an
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endogenous PLR gene (Bayindir et al. 2008). All these earlier studies used genes originating in plants for the metabolic engineering of lignan biosynthesis. In this study, we aimed to assess the possibility of lignan manipulation by the overexpression of a bacterial gene for PrR, which exhibits high catalytic function toward Pin, a key intermediate for a wide variety of lignan compounds.
Materials and methods Plasmid constructions and transformation of A. thaliana The nucleotide sequences of the Sphingobium sp. strain SYK6 (NBRC 103272) chromosome and pinZ were deposited in the DDBJ/EMBL/GenBank databases with the accession number AP012222 and the locus tag SLG_07320, respectively. A pinZ complementary DNA (cDNA) (mpinZ) with optimized codon usage for expression in A. thaliana was chemically synthesized with a DNA synthesizer without changing the amino acid sequence of the PinZ polypeptide from the original genomic sequence of the gene and cloned in pIDTSMART (Fig. S1 in the Supplementary Material). The sequence of mpinZ is deposited in the databases with the accession number AB924082. An expression cassette of pinZ was constructed in a binary vector pBF2 under the control of the CaMV35S promoter (Fig. 2a). The resultant vector was then transferred into Agrobacterium tumefaciens GV3101 by electroporation. A. thaliana was transformed by the floral dip procedure using the recombinant A. tumefaciens. Seeds from the inoculated plants were recovered, and candidates of the transgenic plant were selected on Murashige and Skoog (MS) medium supplemented with 50 μg/mL kanamycin. Stable homozygous transformants with the pinZ transgene were selected by several rounds of kanamycin selection with seeds of independent T1 and T2 generations. Growth conditions of plants Seeds from the wild-type and transgenic plants were surfacesterilized with 5 % sodium hypochlorite and 0.3 % Tween-20 and sown on a 0.8 % (w/v) agar-solidified medium supplemented with MS salt, 3 % (w/v) sucrose, 100 mg/L myoinositol, 2 mg/L L-glycine, 0.5 mg/L nicotinic acid, 0.5 mg/ L pyridoxine hydrochloride, and 0.1 mg/L thiamine hydrochloride. The seeds were incubated for 2–3 days at 4 °C and then placed under a 16-h light/8-h dark regime at 23 °C for approximately 2 weeks. For the preparation of root samples (4 weeks old), ten individual plants were transferred to fresh MS medium in rectangular plates, set perpendicularly, and incubated for a further 2 weeks under identical conditions. For the preparation of stem and leaf samples (6 weeks old), the seedlings were
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transferred to fresh MS medium and incubated for a further 2 weeks after the first 2-week incubation on MS medium. Subsequently, individual plants were planted (vermiculite/ perlite/peat moss=2:1:1, v/v) in polypropylene pots and grown for 2 weeks. Preparation of crude enzyme and detection of PinZ activity Seedling, root, leaf, and stem samples were ground in liquid nitrogen, and crude enzyme was extracted with 500 μL of 50 mM Tris-HCl (pH 7.5), followed by centrifugation at 15,000 rpm for 10 min at 4 °C. The supernatants including the crude enzyme were purified twice by centrifugation at 15,000 rpm for 10 min at 4 °C. Protein was quantified by the Bradford method (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories Inc., Hercules, CA, USA) with bovine serum albumin as a standard protein. A 5- (low concentration) or 50-μg (high concentration) sample of the crude enzyme was incubated with 100 μM (±)-Pin and 500 μM β-NADPH in 500 μL of 50 mM Tris–HCl (pH 7.5) for 1, 3, 5, and 10 min and 24 h. The reaction mixture was extracted thrice with 2 mL of ethyl acetate, and the resulting mixture was centrifuged to separate the organic and aqueous layers at 5,000 rpm for 10 min. The organic layers of supernatants were collected, pooled, and evaporated. The precipitate was dissolved with the solvent used for mobile phase, described below. The reaction mixture was analyzed with high- and ultraperformance liquid chromatography (HPLC and UPLC) equipment coupled with a photodiode array (PDA) and with PDA and mass spectrometer (MS), respectively, as detectors. The HPLC system used in this study was a Hitachi LaChrom Elite with a SenshuPak ODS-1251-SS column (4.6×250 mm) at 40 °C. The reaction product was dissolved in 40 % MeOH, and the solution was filtered through a 0.45-μm syringe filter (EMD Millipore Corporation, Billerica, MA, USA). The mobile phase consisted of 40 % methanol at a flow rate of 0.5 mL/min, and the chromatogram was recorded at 280 nm. The PinZ activity was expressed as the rate of total Lar production from Pin with 1 mg of crude enzyme from the tissue in a 1-min reaction time. Identification of the reaction products was performed by UPLC (ACQUITY UPLC system; Nihon Waters K.K., Tokyo, Japan) coupled with a PDA and with an ACQUITY TQ (MS) detector (Waters) using a TSKgel ODS-140HTP column (2.1×100 mm; Tosoh Corporation, Tokyo, Japan). The authentic compounds (±)-Pin, (±)-Lar, and (±)-Sec used in this analysis were chemically synthesized as described in our previous report (Fukuhara et al. 2013). The precipitated reaction mixture was dissolved in 400 μL of a mixture of water and acetonitrile (75:25, v/v). The mobile phase was a mixture of water (75%) and acetonitrile (25%) containing 0.1 % formic acid, and the flow rate was 0.3 mL/min. Mass spectra were obtained in negative and positive modes with the
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following settings: capillary voltage, 3.0 kV; cone voltage, 10–40 V; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow rate, 650 L/h; and cone gas flow rate, 50 L/h. Chiral HPLC analysis was performed to determine the qualitative enantiomeric compositions of pinoresinol in the reaction mixtures with 20 μg of the crude enzymes from the wild type, the transgenic (line PinZ8) plants, and recombinant Escherichia coli BL21(DE3) harboring pinZ (Fukuhara et al. 2013). Reaction products prepared as described above, and the authentic compounds (racemic pinoresinol and lariciresinol) were dissolved in hexane/ethanol (3:2, v/v) and then separated by an HPLC system (LC-2000, JASCO Corporation, Osaka, Japan) equipped with a Daicel Chiralcel OD-H column (4.6 mm×250 mm, Daicel Corporation, Osaka, Japan) and a UV detector (UV-2075, JASCO Corporation) with a flow rate of 1.5 mL/min of hexane/ethanol (1:1, v/v). Expression analysis by sqRT-PCR Root and stem samples were ground in liquid nitrogen, and total RNA was then extracted with 1 mL of TRIzol® reagent (Life Technologies Japan Ltd., Tokyo, Japan). After the samples were refined twice with a solution of 2 M LiCl, RNA was quantified by spectrophotometry (JASCO Inc., Easton, MD, USA). A 1-μg aliquot of total RNA was used to synthesize first-strand complementary DNA using the Transcriptor first strand cDNA synthesis kit (Roche Diagnostics K.K., Tokyo, Japan). To confirm the presence of transcripts, semiquantitative reverse transcription-polymerase chain reaction (sqRTPCR) was performed with the following gene-specific primers (Table S1 in the Supplementary Material). Amplification products were separated on a 1.5 % agarose gel and stained with 1 μg/mL ethidium bromide. Western blot analysis The crude enzymes (5 μg) prepared from the seven independent transgenic and wild-type plants were subjected to sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDSPAGE) and subsequent Western blot analysis. SDS-PAGE was performed on 10 % polyacrylamide gels. The histidinetagged PinZ, whose estimated molecular weight is 36,926, was also used as positive control. After the SDS-PAGE, the separated enzymes were transferred to a polyvinylidene difluoride membrane in a semidry blotting system. After blocking with a 3 % (w/v) solution of skim milk for 1 h, the blot was incubated for 1 h with diluted-rabbit anti-serum raised against an oligopeptide, RYGDYDKPETLADAC, corresponding to amino acids 54 to 67 of the PinZ polypeptide (the estimated molecular weight of PinZ is 34,316). Peroxidase-conjugated antibodies, raised in goat against rabbit immunoglobulin, were then incubated with the blot for 1 h,
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and the PinZ polypeptide was detected by chemiluminescence equipped with AE-9300 Ez-Capture MG (Atto Corporation, Tokyo). Recombinant protein of PinZ with a histidine tag produced in E. coli was used as a positive control (Fukuhara et al. 2013). Untargeted metabolomic analysis Using wild-type and two transgenic lines, PinZ6 and PinZ8, untargeted metabolomic profiling was performed to reveal the influence of pinZ expression in the plants. Extracts from dried roots, leaves, and stems of the plants grown under conditions as described above were analyzed separately using six independent biological replicates for each line. The dried samples of the tissues (2 mg each) were extracted with 50 μL of 80 % MeOH containing 2.5 μM lidocaine and 10-camphorsulfonic acid per milligram dry weight using a mixer mill with zirconia beads for 7 min at 18 Hz and 4 °C. After centrifugation for 10 min, the supernatant was filtered using an HLB μElution plate (Waters). The extracts (1 μL) were analyzed using LC-quadrupole time-of-flight (QTOF)-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). Analytical conditions were as follows: column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 μm, 2.1 mm×100 mm, Waters); solvent system, solvent A (water including 0.1 % formic acid) and solvent B (acetonitrile including 0.1 % formic acid); gradient program, 99.5 % A/0.5 % B at 0 min, 99.5 % A/0.5 % B at 0.1 min, 20 % A/80 % B at 10 min, 0.5 % A/99.5 % B at 10.1 min, 0.5 %A/ 99.5 %B at 12.0 min, 99.5 % A/0.5 % B at 12.1 min, and 99.5 % A/0.5 % B at 15.0 min; flow rate, 0.3 mL/min at 0 min, 0.3 mL/min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min, and 0.3 mL/min at 14.5 min; and column temperature, 40 °C. The analytical conditions for MS detection were as follows: capillary voltage, +3.0 keV; cone voltage, 25.0 V; source temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow, 50 L/h; desolvation gas flow, 800 L/h; collision energy, 6 V; mass range, m/z 100–1,500; scan duration, 0.1 s; interscan delay, 0.014 s; data acquisition, centroid mode; polarity, positive/negative; lockspray (leucine enkephalin) scan duration, 1.0 s; and interscan delay, 0.1 s. The MS/MS data were acquired in ramp mode under the following analytical conditions: (1) MS: mass range, m/z 50–1,500; scan duration, 0.1 s; interscan delay, 0.014 s; and data acquisition, centroid mode and (2) MS/MS: mass range, m/z 50–1,500; scan duration, 0.02 s; interscan delay, 0.014 s; data acquisition, centroid mode; and polarity, positive/negative collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra for the top 10 ions (>1,000 counts) in an MS scan were automatically obtained. If the ion intensity was
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Successful expression of a novel bacterial gene for pinoresinol reductase and its effect on lignan biosynthesis in transgenic Arabidopsis thaliana Masayuki Tamura & Yukiko Tsuji & Tatsuya Kusunose & Atsushi Okazawa & Naofumi Kamimura & Tetsuya Mori & Ryo Nakabayashi & Shojiro Hishiyama & Yuki Fukuhara & Hirofumi Hara & Kanna Sato-Izawa & Toshiya Muranaka & Kazuki Saito & Yoshihiro Katayama & Masao Fukuda & Eiji Masai & Shinya Kajita
Received: 8 April 2014 / Revised: 2 July 2014 / Accepted: 5 July 2014 / Published online: 24 July 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Pinoresinol reductase and pinoresinol/lariciresinol reductase play important roles in an early step of lignan biosynthesis in plants. The activities of both enzymes have also been detected in bacteria. In this study, pinZ, which was first isolated as a gene for bacterial pinoresinol reductase, was constitutively expressed in Arabidopsis thaliana under the control of the cauliflower mosaic virus 35S promoter. Higher reductive activity toward pinoresinol was detected in the resultant transgenic plants but not in wild-type plant. Principal component analysis of data from untargeted metabolome analyses of stem, root, and leaf extracts of the wild-type
and two independent transgenic lines indicate that pinZ expression caused dynamic metabolic changes in stems, but not in roots and leaves. The metabolome data also suggest that expression of pinZ influenced the metabolisms of lignan and glucosinolates but not so much of neolignans such as guaiacylglycerol-8-O-4′-feruloyl ethers. In-depth quantitative analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS) indicated that amounts of pinoresinol and its glucoside form were markedly reduced in the transgenic plant, whereas the amounts of glucoside form of secoisolariciresinol in transgenic roots, leaves, and stems increased. The detected
Electronic supplementary material The online version of this article (doi:10.1007/s00253-014-5934-x) contains supplementary material, which is available to authorized users. M. Tamura : Y. Tsuji : K. Sato-Izawa : S. Kajita (*) Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, 2-24-16 Naka-cho, Koganei, Tokyo 184-8588, Japan e-mail: [email protected] T. Kusunose : A. Okazawa : T. Muranaka Graduate School of Engineering, Osaka University, 1-1 Yamadaoka, Suita, Osaka 565-0871, Japan N. Kamimura : Y. Fukuhara : M. Fukuda : E. Masai Department of Bioengineering, Nagaoka University of Technology, 1603-1 Kamitomioka, Nagaoka, Niigata 940-2188, Japan T. Mori : R. Nakabayashi : K. Saito Metabolomics Research Group, RIKEN Center for Sustainable Resource Science, RIKEN, 1-7-22, Tsurumi, Kanagawa 230-0045, Japan S. Hishiyama Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki 305-8687, Japan
H. Hara Department of Environmental Engineering and Green Technology, Malaysia-Japan International Institute of Technology, Universiti Teknologi Malaysia, Kuala Lumpur 54100, Malaysia
K. Saito Graduate School of Pharmaceutical Sciences, Chiba University, 1-8-1 Inohana, Chuo-ku, Chiba 260-8675, Japan
Y. Katayama College of Bioresource Sciences, Nihon University, 1866 Mameino, Fujisawa, Kanagawa 252-0880, Japan Present Address: A. Okazawa Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, NakakuSakai Osaka 5998531, Japan
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levels of lariciresinol in the transgenic plant following βglucosidase treatment also tended to be higher than those in the wild-type plant. Our findings indicate that overexpression of pinZ induces change in lignan compositions and has a major effect not only on lignan biosynthesis but also on biosynthesis of other primary and secondary metabolites.
et al. 2013; Suzuki and Umezawa 2007). Most lignans are biosynthesized enantiospecifically. For example, in Forsythia intermedia, two molecules of coniferyl alcohol are dehydrogenated by an oxidase such as laccase to give two coniferyl alcohol radicals, and then, they are coupled under the control of the dirigent protein, which plays the important role of structural regulator, and (+)-Pin is preferentially generated (Davin et al. 1997). (+)-Pin is further converted to (−)secoisolariciresinol (Sec) through (+)-lariciresinol (Lar) in a reaction catalyzed by pinoresinol/lariciresinol reductase (PLR; Dinkova-Kostova et al. 1996). In Arabidopsis thaliana, pinoresinol reductase 1 (AtPrR1) can reduce both (+)- and (−)Pin to generate (+)- and (−)-Lar (Nakatsubo et al. 2008). Lignans exhibit a broad range of biological activities, including anti-viral, anti-fungal, anti-bacterial, and antioxidant activity, root elongation inhibition, and anti-feedant activity against insects (Deyama and Nishibe 2010). Although these activities suggest that lignans are important for plant protection against biotic and abiotic stresses, most are still under investigation (Harmatha and Dinan 2003; Lorenc-Kukuła et al. 2009). Lignans are also characterized as compounds with a variety of pharmaceutical applications. For example, the seeds of flax contain high levels of secoisolariciresinol diglucoside (SDG), which displays a wide range of health-
Keywords Arabidopsis thaliana . Lignan . Pinoresinol reductase . Sphingobium sp. SYK-6
Introduction Lignans are common secondary metabolites in plants. It is synthesized through the coupling of phenylpropanoid monomers such as coniferyl alcohol, linked through the 8 and 8′ carbons in side chains of the monomers. A large variety of lignans with different chemical structures have been isolated from plants, and they are categorized based on their structure such as furan, furofuran, and dibenzylbutane. Among the wide range of lignans, molecules synthesized through pinoresinol (Pin) have been structurally characterized, and their biosynthetic pathways have been extensively investigated (Fig. 1; Davin and Lewis 2003; Davin et al. 2008; Fuss 2003; Satake
Neolignans 7′
6′ 9 7
Derivatives
1′
5′
8′
8
9′
Lignans
2′
4′
3′
1 6
2
5
3
f eruloyl ether
4
?
Aromatic Amino Acid
Lariciresinol monoglucoside (Lar-Glc)
Dehydrodiconiferyl alcohol
?
3 2
4
9 1 8 7
PinZ AtPrR1
8 8′
2 3
5
PinZ
AtPrR1, AtPrR2
6
9′ 1
6
5
7
9
6′ 1′
5′
4 4′
2′ 3′
7′
Lariciresinol (Lar)
Pinoresinol (Pin)
Secoisolariciresinol (Sec)
Conif eryl alcohol
Ferulic acid
?
Feruloyl-CoA
Conif eraldehyde
Epipinoresinol (Epi)
Syringaresinol gucosides
PinZ Syringaresinol Sinapic acid
Secoisolariciresinol diglucosides (SDG)
Matairesinol (Mat)
Dimethoxylariciresinol glucosides Dimethoxylariciresinol
Sinapyl alcohol
Fig. 1 The predicted biosynthetic pathway of lignans, neolignans, and related compounds in Arabidopsis thaliana and catalytic steps catalyzed by PinZ. AtPrR1 and AtPrR2 are endogenous pinoresinol reductases of A. thaliana
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promoting activities including radical-scavenging and anticancer effects (Lainé et al. 2009; Prasad 1997). In addition, podophyllotoxin, isolated from Podophyllum plants, is a precursor used to produce drugs targeting testicular cancer and small cell lung carcinoma (Damayanthi and Lown 1998). Lignans, including Pin, Lar, and Sec, are thought to be metabolized by mammalian intestinal bacteria to produce enterodiol and enterolactone, which modulate immune responses and have been classified as mammalian lignans (Heinonen et al. 2001; Kurzer and Xu 1997; Landete 2012; Parr and Bolwell 2000). Structural transformations of lignan compounds by intestinal bacteria have been characterized, and some enzymatic functions are known. For example, Enterococcus faecalis strain PDG-1 can convert (+)-Pin to (+)-Lar, and both Pin and Lar are transformed to Sec by Eggerthella lenta (Clavel et al. 2006; Xie et al. 2003). These observations suggest that intestinal bacteria possess identical types of catabolic enzymes for lignan transformation as do plants. However, few catabolic genes for these lignan transformations had been cloned from bacteria, including intestinal bacteria. Recently, we successfully cloned two genes, pinZ and Saro_2808, for pinoresinol reductase (PrR) from the bacteria Sphingobium sp. SYK-6 and Novosphingobium aromaticivorans DSM 1244, respectively (Fukuhara et al. 2013). This was the first report of the cloning of bacterial PrR genes. Although the deduced amino acid sequence of pinZ shares no significant identity with that of plant-origin PrR or PLR, recombinant PinZ catalyzed the reduction of (±)Pin to (±)-Lar and of (±)-syringaresinol to (±)dimethoxylariciresinol in the presence of NADPH (Fukuhara et al. 2013). In addition, the specific activity of recombinant PinZ toward (±)-Pin was over 1,000-fold higher than that of recombinant AtPrR1, reported by Nakatsubo et al. (2008). PinZ also has Lar-reducing activity, although this activity is weaker than a similar activity toward Pin (Fukuhara et al. 2013). In the present study, pinZ was overexpressed in transgenic A. thaliana plants under the control of the cauliflower mosaic virus 35S (CaMV35S) promoter with the aim of modulating lignan accumulation. In previous studies, metabolic engineering of lignan accumulation has already been achieved in some transgenic plants. Ayella et al. (2007) and Banerjee and Chattopadhyay (2010) reported the overaccumulation of SDG and phyllanthin in transgenic wheat and Phyllanthus amarus plants, respectively, overexpressing PLR genes of plant origin. Furthermore, sesamin accumulation has been achieved by a combination of the downregulation of PLR and the overexpression of Sesamum CYP81Q1 genes in transgenic Forsythia cell cultures (Kim et al. 2009). Accumulation of hinokinin, which is synthesized via matairesinol (Mat), was almost completely suppressed in hairy roots of Linum corymbulosum with the RNA interference construct for an
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endogenous PLR gene (Bayindir et al. 2008). All these earlier studies used genes originating in plants for the metabolic engineering of lignan biosynthesis. In this study, we aimed to assess the possibility of lignan manipulation by the overexpression of a bacterial gene for PrR, which exhibits high catalytic function toward Pin, a key intermediate for a wide variety of lignan compounds.
Materials and methods Plasmid constructions and transformation of A. thaliana The nucleotide sequences of the Sphingobium sp. strain SYK6 (NBRC 103272) chromosome and pinZ were deposited in the DDBJ/EMBL/GenBank databases with the accession number AP012222 and the locus tag SLG_07320, respectively. A pinZ complementary DNA (cDNA) (mpinZ) with optimized codon usage for expression in A. thaliana was chemically synthesized with a DNA synthesizer without changing the amino acid sequence of the PinZ polypeptide from the original genomic sequence of the gene and cloned in pIDTSMART (Fig. S1 in the Supplementary Material). The sequence of mpinZ is deposited in the databases with the accession number AB924082. An expression cassette of pinZ was constructed in a binary vector pBF2 under the control of the CaMV35S promoter (Fig. 2a). The resultant vector was then transferred into Agrobacterium tumefaciens GV3101 by electroporation. A. thaliana was transformed by the floral dip procedure using the recombinant A. tumefaciens. Seeds from the inoculated plants were recovered, and candidates of the transgenic plant were selected on Murashige and Skoog (MS) medium supplemented with 50 μg/mL kanamycin. Stable homozygous transformants with the pinZ transgene were selected by several rounds of kanamycin selection with seeds of independent T1 and T2 generations. Growth conditions of plants Seeds from the wild-type and transgenic plants were surfacesterilized with 5 % sodium hypochlorite and 0.3 % Tween-20 and sown on a 0.8 % (w/v) agar-solidified medium supplemented with MS salt, 3 % (w/v) sucrose, 100 mg/L myoinositol, 2 mg/L L-glycine, 0.5 mg/L nicotinic acid, 0.5 mg/ L pyridoxine hydrochloride, and 0.1 mg/L thiamine hydrochloride. The seeds were incubated for 2–3 days at 4 °C and then placed under a 16-h light/8-h dark regime at 23 °C for approximately 2 weeks. For the preparation of root samples (4 weeks old), ten individual plants were transferred to fresh MS medium in rectangular plates, set perpendicularly, and incubated for a further 2 weeks under identical conditions. For the preparation of stem and leaf samples (6 weeks old), the seedlings were
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transferred to fresh MS medium and incubated for a further 2 weeks after the first 2-week incubation on MS medium. Subsequently, individual plants were planted (vermiculite/ perlite/peat moss=2:1:1, v/v) in polypropylene pots and grown for 2 weeks. Preparation of crude enzyme and detection of PinZ activity Seedling, root, leaf, and stem samples were ground in liquid nitrogen, and crude enzyme was extracted with 500 μL of 50 mM Tris-HCl (pH 7.5), followed by centrifugation at 15,000 rpm for 10 min at 4 °C. The supernatants including the crude enzyme were purified twice by centrifugation at 15,000 rpm for 10 min at 4 °C. Protein was quantified by the Bradford method (Bio-Rad Protein Assay Kit, Bio-Rad Laboratories Inc., Hercules, CA, USA) with bovine serum albumin as a standard protein. A 5- (low concentration) or 50-μg (high concentration) sample of the crude enzyme was incubated with 100 μM (±)-Pin and 500 μM β-NADPH in 500 μL of 50 mM Tris–HCl (pH 7.5) for 1, 3, 5, and 10 min and 24 h. The reaction mixture was extracted thrice with 2 mL of ethyl acetate, and the resulting mixture was centrifuged to separate the organic and aqueous layers at 5,000 rpm for 10 min. The organic layers of supernatants were collected, pooled, and evaporated. The precipitate was dissolved with the solvent used for mobile phase, described below. The reaction mixture was analyzed with high- and ultraperformance liquid chromatography (HPLC and UPLC) equipment coupled with a photodiode array (PDA) and with PDA and mass spectrometer (MS), respectively, as detectors. The HPLC system used in this study was a Hitachi LaChrom Elite with a SenshuPak ODS-1251-SS column (4.6×250 mm) at 40 °C. The reaction product was dissolved in 40 % MeOH, and the solution was filtered through a 0.45-μm syringe filter (EMD Millipore Corporation, Billerica, MA, USA). The mobile phase consisted of 40 % methanol at a flow rate of 0.5 mL/min, and the chromatogram was recorded at 280 nm. The PinZ activity was expressed as the rate of total Lar production from Pin with 1 mg of crude enzyme from the tissue in a 1-min reaction time. Identification of the reaction products was performed by UPLC (ACQUITY UPLC system; Nihon Waters K.K., Tokyo, Japan) coupled with a PDA and with an ACQUITY TQ (MS) detector (Waters) using a TSKgel ODS-140HTP column (2.1×100 mm; Tosoh Corporation, Tokyo, Japan). The authentic compounds (±)-Pin, (±)-Lar, and (±)-Sec used in this analysis were chemically synthesized as described in our previous report (Fukuhara et al. 2013). The precipitated reaction mixture was dissolved in 400 μL of a mixture of water and acetonitrile (75:25, v/v). The mobile phase was a mixture of water (75%) and acetonitrile (25%) containing 0.1 % formic acid, and the flow rate was 0.3 mL/min. Mass spectra were obtained in negative and positive modes with the
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following settings: capillary voltage, 3.0 kV; cone voltage, 10–40 V; source temperature, 120 °C; desolvation temperature, 350 °C; desolvation gas flow rate, 650 L/h; and cone gas flow rate, 50 L/h. Chiral HPLC analysis was performed to determine the qualitative enantiomeric compositions of pinoresinol in the reaction mixtures with 20 μg of the crude enzymes from the wild type, the transgenic (line PinZ8) plants, and recombinant Escherichia coli BL21(DE3) harboring pinZ (Fukuhara et al. 2013). Reaction products prepared as described above, and the authentic compounds (racemic pinoresinol and lariciresinol) were dissolved in hexane/ethanol (3:2, v/v) and then separated by an HPLC system (LC-2000, JASCO Corporation, Osaka, Japan) equipped with a Daicel Chiralcel OD-H column (4.6 mm×250 mm, Daicel Corporation, Osaka, Japan) and a UV detector (UV-2075, JASCO Corporation) with a flow rate of 1.5 mL/min of hexane/ethanol (1:1, v/v). Expression analysis by sqRT-PCR Root and stem samples were ground in liquid nitrogen, and total RNA was then extracted with 1 mL of TRIzol® reagent (Life Technologies Japan Ltd., Tokyo, Japan). After the samples were refined twice with a solution of 2 M LiCl, RNA was quantified by spectrophotometry (JASCO Inc., Easton, MD, USA). A 1-μg aliquot of total RNA was used to synthesize first-strand complementary DNA using the Transcriptor first strand cDNA synthesis kit (Roche Diagnostics K.K., Tokyo, Japan). To confirm the presence of transcripts, semiquantitative reverse transcription-polymerase chain reaction (sqRTPCR) was performed with the following gene-specific primers (Table S1 in the Supplementary Material). Amplification products were separated on a 1.5 % agarose gel and stained with 1 μg/mL ethidium bromide. Western blot analysis The crude enzymes (5 μg) prepared from the seven independent transgenic and wild-type plants were subjected to sodium dodecyl sulfate-polyacylamide gel electrophoresis (SDSPAGE) and subsequent Western blot analysis. SDS-PAGE was performed on 10 % polyacrylamide gels. The histidinetagged PinZ, whose estimated molecular weight is 36,926, was also used as positive control. After the SDS-PAGE, the separated enzymes were transferred to a polyvinylidene difluoride membrane in a semidry blotting system. After blocking with a 3 % (w/v) solution of skim milk for 1 h, the blot was incubated for 1 h with diluted-rabbit anti-serum raised against an oligopeptide, RYGDYDKPETLADAC, corresponding to amino acids 54 to 67 of the PinZ polypeptide (the estimated molecular weight of PinZ is 34,316). Peroxidase-conjugated antibodies, raised in goat against rabbit immunoglobulin, were then incubated with the blot for 1 h,
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and the PinZ polypeptide was detected by chemiluminescence equipped with AE-9300 Ez-Capture MG (Atto Corporation, Tokyo). Recombinant protein of PinZ with a histidine tag produced in E. coli was used as a positive control (Fukuhara et al. 2013). Untargeted metabolomic analysis Using wild-type and two transgenic lines, PinZ6 and PinZ8, untargeted metabolomic profiling was performed to reveal the influence of pinZ expression in the plants. Extracts from dried roots, leaves, and stems of the plants grown under conditions as described above were analyzed separately using six independent biological replicates for each line. The dried samples of the tissues (2 mg each) were extracted with 50 μL of 80 % MeOH containing 2.5 μM lidocaine and 10-camphorsulfonic acid per milligram dry weight using a mixer mill with zirconia beads for 7 min at 18 Hz and 4 °C. After centrifugation for 10 min, the supernatant was filtered using an HLB μElution plate (Waters). The extracts (1 μL) were analyzed using LC-quadrupole time-of-flight (QTOF)-MS (LC, Waters Acquity UPLC system; MS, Waters Xevo G2 Q-Tof). Analytical conditions were as follows: column, Acquity bridged ethyl hybrid (BEH) C18 (1.7 μm, 2.1 mm×100 mm, Waters); solvent system, solvent A (water including 0.1 % formic acid) and solvent B (acetonitrile including 0.1 % formic acid); gradient program, 99.5 % A/0.5 % B at 0 min, 99.5 % A/0.5 % B at 0.1 min, 20 % A/80 % B at 10 min, 0.5 % A/99.5 % B at 10.1 min, 0.5 %A/ 99.5 %B at 12.0 min, 99.5 % A/0.5 % B at 12.1 min, and 99.5 % A/0.5 % B at 15.0 min; flow rate, 0.3 mL/min at 0 min, 0.3 mL/min at 10 min, 0.4 mL/min at 10.1 min, 0.4 mL/min at 14.4 min, and 0.3 mL/min at 14.5 min; and column temperature, 40 °C. The analytical conditions for MS detection were as follows: capillary voltage, +3.0 keV; cone voltage, 25.0 V; source temperature, 120 °C; desolvation temperature, 450 °C; cone gas flow, 50 L/h; desolvation gas flow, 800 L/h; collision energy, 6 V; mass range, m/z 100–1,500; scan duration, 0.1 s; interscan delay, 0.014 s; data acquisition, centroid mode; polarity, positive/negative; lockspray (leucine enkephalin) scan duration, 1.0 s; and interscan delay, 0.1 s. The MS/MS data were acquired in ramp mode under the following analytical conditions: (1) MS: mass range, m/z 50–1,500; scan duration, 0.1 s; interscan delay, 0.014 s; and data acquisition, centroid mode and (2) MS/MS: mass range, m/z 50–1,500; scan duration, 0.02 s; interscan delay, 0.014 s; data acquisition, centroid mode; and polarity, positive/negative collision energy, ramped from 10 to 50 V. In this mode, MS/MS spectra for the top 10 ions (>1,000 counts) in an MS scan were automatically obtained. If the ion intensity was
