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Hyper-production of 13C-labeled trans-resveratrol in Vitis vinifera suspension cell culture by elicitation and in situ adsorption
Biochemical Engineering Journal 53 (2011) 292–296
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Hyper-production of 13 C-labeled trans-resveratrol in Vitis vinifera suspension cell culture by elicitation and in situ adsorption Xiangguo Yue a,b , Wei Zhang a,c,∗ , Maicun Deng b a b c
Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Graduate School of the Chinese Academy of Sciences, Beijing 100039, China Flinders Centre for Marine Bioprocessing and Bioproducts, and Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide, SA 5042, Australia
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Article history: Received 6 July 2010 Received in revised form 9 December 2010 Accepted 12 December 2010 Available online 21 December 2010 Keywords: Vitis vinifera 13 C-labeled trans-resveratrol Secondary metabolites Plant cell tissue Culture engineering Metabolic control Adsorption
a b s t r a c t Bioproduction of 13 C-labeled trans-resveratrol in plant cell culture has attracted considerable attention with regard to potential applications for human benefit and to better understanding their absorption and in vivo metabolism in humans and animals. In the present work, two elicitors (SA and JA) and adsorbents (HP2MGL) were introduced into the Vitis vinifera cell suspension culture, and the results indicated that they could work synergistically to improve the production of trans-resveratrol (2666.7 mg L−1 ). Afterward, 1 mM [1-13 C]-l-phenylalanine (Phe) was added to bioproduce 13 C-labeled trans-resveratrol with the 13 C enrichment from 35.7% at day 5 to 20.8% at day 10. Purification of the products by several chromatographic steps was reported, and the 13 C labeling position was verified using 13 C NMR. Our results indicated that, in this case, the rate limiting step of production was the post-biosynthetic events rather than the biosynthesis in elicited culture. Furthermore, our results suggested the importance of simultaneous optimization of biosynthetic pathways of metabolites and their post-biosynthetic steps toward achieving commercial plant cell culture. With the help of this technique, we could expect to do industrial scaling-up in a bioreactor to produce high amounts of the non-labeled and 13 C-labeled trans-resveratrol in the near future. © 2011 Elsevier B.V. All rights reserved.
1. Introduction trans-Resveratrol (3,5,4 -trihydroxystilbene) is a natural compound belonging to the stilbene family. It is produced in response to biotic and abiotic stress, therefore acting like a phytoalexin [1,2]. It was first isolated from white hellebore [3] and has been reported in several plants, such as peanut, lily, mulberries, eucalyptus, spruce and pine [4–6]. However, a Chinese herbal medicinal plant, Polygonum cuspidatum and grapevine are currently the main sources of this compound. Resveratrol has become the focus of a number of studies in medicine and plant physiology, and has emerged as a promising molecule that potentially plays a key role in human health [7–15]. For these reasons, the Vitis vinifera cell suspension culture has been studied as a reliable alternative approach to produce isotopically 13 C-labeled trans-resveratrol for investigation of its absorption and the in vivo metabolism in humans and animals [9,16,17]. Various strategies have been established for the production of 13 C-labeled trans-resveratrol with V. vinifera cell culture. Krisa et al.
∗ Corresponding author at: Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. E-mail address: [email protected] (W. Zhang). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.12.002
reported that the total piceid reached 770 mol L−1 with the 13 C enrichment rounded to about 66% at day 14, and the piceid can be extracted from cells and transformed to resveratrol by enzymatic hydrolysis [18]. Subsequently, Aumont et al. used a 2 L stirred-tank bioreactor for producing isotopically 13 C-labeled phenolic substances, in which the piceid production reached 360 mg L−1 [16]. However, there is no report of a cell culture system to produce directly 13 C-labeled resveratrol at high productivity. Previously, there have been a number of reports using synthetic adsorbents to remove products in situ [19–21] and using elicitors to improve resveratrol production [18,22,23]. A Spanish research group has achieved a significantly high resveratrol production in grapevine cell culture by combining elicitation and in situ product removal. Cyclodextrins acted as both elicitors and artificial storage in this experiment for product removal [24]. In our previous studies, different types of absorbents and several elicitors were screened and tested, with the most efficient elicitors and absorbents identified for enhancing the trans-resveratrol production (unpublished results). Additionally, 500 M salicylic acid (SA) was observed to increase extracellular phenolics in combination with 10 M jasmonic acid (JA), despite its poor cell growth (unpublished results). As a result, this project aimed to simultaneously use elicitors (SA and JA) and adsorbents for improving the production of 13 C-labeled resveratrol in the V. vinifera cell culture.
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Moreover, this project was also established to test the hypothesis, in which Zhang et al. suggested the importance of simultaneous optimization of biosynthetic pathways of secondary metabolites and their post-biosynthetic steps toward achieving commercial plant cell culture [25]. The production of 13 C labeled trans-resveratrol was achieved by feeding the [1-13 C]-l-Phe, and the 13 C labeling position of 13 C-labeled trans-resveratrol was verified by the 13 C NMR. 2. Materials and methods 2.1. Chemicals [1-13 C]-l-Phe was purchased from Cambridge Isotope Laboratories, Inc. (USA). trans-Resveratrol and trans-piceid, jasmonic acid (JA), salicylic acid (SA) and l-Phe standards were from Sigma (China). HP2MGL was purchased from Rohm and Hass Company (China). All other chemicals used were of analytical grade, and solvents were purchased in HPLC grade. 2.2. V. vinifera cell suspension cultures The cell line used in this study was developed by Cormier et al. [26], originating from a callus established in 1978 from V. vinifera L. cv. Gamay Fréaux var. teinturier berry pulp. This cell line, capable of anthocyanin and trans-piceid accumulation in the dark, was a gift from Dr. Francois Cormier’s group (Quebec, Canada). The cell line has been subcultured in our lab for over 10 years, and subjected to intensive screening of highly pigmented line. The subcultures were performed by inoculating approximately 2.5 g wet cells into 20 mL of the B5 medium, supplemented with 30 g L−1 sucrose, 250 mg L−1 casein hydrolysate, 0.1 mg L−1 ␣-naphthaleneacetic acid (NAA) and 0.2 mg L−1 kinetin. The wet cells were prepared by filtering precultured 7-day-old suspension cells with a 50-m mesh. The subcultures were conducted weekly in the dark, in 500-mL Erlenmeyer flasks, enclosed with an aluminum foil and were maintained on a reciprocating shaker at 100 rpm and 25 ± 1 ◦ C. 2.3. Pre-treatment of the HP2MGL adsorbent The HP2MGL adsorbent, a non-aromatic resin, based on methacrylic ester copolymer, was first rinsed in Milli-Q water using a 1 L measuring cylinder and then transferred into a 1 L glass column (2.5 cm diameter × 40 cm length). The HP2MGL adsorbent was washed with five bed volumes of 100% methanol at a flow rate of two bed volumes per hour. After this, the adsorbent was washed with ten bed volumes of Milli-Q water at a flow rate of five bed volumes per hour. Finally, the HP2MGL adsorbent was vacuum-filtered and ready for application at the beginning of culture. 2.4. Elicitors treatment and precursor feeding Jasmonic acid (JA) and salicylic acid (SA) were dissolved, respectively, in ethanol–water (12:13) and 100% ethanol. l-Phe and [1-13 C]-l-Phe were prepared according to the method of Krisa et al. [18]. Control cultures received the corresponding vehicle solvent at a final concentration that did not exceed 0.2% (ETOH) or 0.5% (DMSO). SA, JA and Phe were filter-sterilized and added into culture at the beginning of the exponential growth phase (day 4). 2.5. Quantification and identification of anthocyanins and stilbenes (trans-resveratrol and trans-piceid) At the time of sampling, the control cultures were harvested by vacuum filtration through filter paper, washed with Milli-Q water and weighed to obtain the fresh cell weight. To separate cells from
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adsorbents in the treatments, 20% sucrose solution was added, and owing to their different densities, the cells can be collected from the top of the medium whereas the adsorbents congregate on the bottom of Erlenmeyer flasks. The anthocyanin existed only in cells and the content was analyzed according to the protocol reported by Zhang et al. [27]. Stilbenes (trans-piceid or trans-resveratrol) were extracted with 20-fold (v/w) MeOH containing 1% HCl and MeOH from cells and adsorbents, respectively. The mixture was incubated overnight at room temperature and then centrifuged for 10 min at 14,000 rpm. The supernatant was obtained for HPLC analysis. The stilbenes (trans-resveratrol and trans-piceid) extracted from cells and adsorbents were identified by co-elution with an authentic standard. Stilbene contents were estimated from a standard curve that was prepared with standards of trans-resveratrol and trans-piceid. 2.6. Purification of 13 C-labeled trans-resveratrol and measurement of 13 C-trans-resveratrol enrichment To purify the trans-resveratrol, the MeOH extracts of adsorbents were evaporated to dryness and the residue was re-dissolved in 5 mL MeOH. The solutions were purified on a reversed-phase C18 column. The fractions containing trans-resveratrol were condensed and purified through a silica gel column followed by elution with methanol and water. Finally, the obtained extracts were purified by semi-preparative HPLC on a SEP-PAK C18 column. The solution containing trans-resveratrol was filtered and vacuum dried to produce a white solid powder, i.e. trans-resveratrol. The above chromatogram operations were all monitored at 286 and 307 nm. The position of 13 C labeling was verified by NMR (Varian INOVA 400 M NMR). The percentage of 13 C enrichment was measured by EA-IRMS (Flash EA 1112 HT, Finnigan MAT Delta V advantage). Samples were injected via an automatic injector into the elemental analyzer and totally converted to CO2 . The CO2 was transferred to the IRMS using helium (120 ml min−1 ). The different isotopomers were separated by a uniform magnetic field and collected in three different collectors at m/z 44, 45 and 46. A known percentage enrichment of [1-13 C]-l-phenylalanine (99%) was regularly analyzed to verify the validity of the method. 13 C atom percentage was transformed to 13 C enrichment percentage with the formula 13 C enrichment (%) = 13 C atom (%) × number of C per molecule [16,18]. 3. Results 3.1. Growth, anthocyanin and stilbenes accumulation of V. vinifera cell suspension culture To understand the growth, anthocyanin and stilbene accumulation in grape cells for determining the optimal period for stilbene production, cultures were analyzed throughout a 21-day period (Fig. 1). Maximal production of anthocyanins and trans-piceid (about 175 and 440 mg L−1 , respectively) occurred on days 17 and 14, respectively. The maximum cell growth of about 17 g DCW L−1 was reached on day 10. In addition, the medium was examined, and either little or no anthocyanin and stilbene was detected (unpublished data). 3.2. Effect of simultaneous elicitation and addition of adsorbents Based on our previous lab results, elicitors (JA 10 M, SA 500 M added on day 4) and HP2MGL (200 g L−1 added at the first day) were introduced to enhance the production of transresveratrol (Fig. 2). The results demonstrated that the cell growth, as well as the production of anthocyanin and stilbenes, were suppressed deleteriously when JA and/or SA was added in the
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Fig. 1. Time course of cell growth, anthocyanin and trans-piceid biosynthesis in Vitis vinifera cell suspension culture. Data represents the mean ± S.D. of three replicates.
Fig. 2. Effect of the addition of JA, SA and HP2MGL on cell growth, anthocyanin, trans-piceid and trans-resveratrol production on the V. vinifera cell suspension culture. JA (10 M) and SA (500 M) were added on day 4. HP2MGL (200 g L−1 ) was added at the first day, and cells and adsorbents were harvested on day 10. Data are the mean ± S.D. of three replicates.
absence of HP2MGL. In the presence of HP2MGL, 1826.7 mg L−1 trans-resveratrol was produced, with a significant decrease in anthocyanin production (from 100 mg L−1 to 42.3 mg L−1 ). However, the cell growth (10.6 DCW L−1 ) was comparable to the control (12.2 DCW L−1 ). When the elicitors and adsorbents were introduced simultaneously, the total production of trans-resveratrol was further improved, with 2666.7 mg L−1 achieved in the presence of adsorbent and both elicitors. The growth and anthocyanin production were suppressed to some extent (4.2 DCW L−1 , and 33.5 mg L−1 , respectively). In all experiments, the trans-resveratrol is secreted into medium and adsorbed in adsorbents, with minor or undetectable level in the cells and medium, while trans-piceid and anthocyanins are accumulated only in the cells. 3.3. Addition of l-Phe in the presence of elicitation and adsorbents The effect of different feeding time points and concentrations of l-Phe on the production of trans-resveratrol was examined (Fig. 3). Three different concentrations (1 mM, 2 mM, and 3 mM) were applied from the beginning to the middle of the exponential phase (days 4–7). The results showed that, regardless of the concentrations, the beginning of exponential phase (day 4) was the best time to maximize the production of trans-resveratrol, similar to the results of Krisa et al. [16,18,23]. Furthermore, addition of
Fig. 3. Effect of feeding time points and concentration of l-Phe on cell growth, anthocyanin and trans-resveratrol production in the V. vinifera cell suspension culture. Different concentrations (1 mM, 2 mM, and 3 mM) l-Phe were added on days 4, 5, 6, 7, respectively. JA (10 M), SA (500 M) and HP2MGL (200 g L−1 ) were added in all the tests except the control. Cells and adsorbents were harvested on day 10. Data are the mean ± S.D. of three replicates.
Fig. 4. Effect of [1-13 C]-l-Phe feeding on cell growth, anthocyanin and transresveratrol production in V. vinifera cell suspension culture, and the 13 C-transresveratrol enrichment. [1-13 C]-l-Phe (1 mM) was added on day 4. JA (10 M), SA (500 M) and HP2MGL (200 g L−1 ) were added in all the tests. Cells and adsorbents were harvested on days 5, 6, 7,8, 9, 10, respectively, Data are the ± S.D. of three replicates
l-Phe (1–10 mM) did not result in a significant increase in production of trans-resveratrol (laboratory data), and l-Phe feeding could slightly suppress the anthocyanin production. The optimal production conditions of 13 C-labeled trans-resveratrol were established as feeding 1 mM [1-13 C]-l-Phe on day 4, when 10 M JA and 500 M SA were added on day 4, with the presence of 200 g L−1 HP2MGL from the first day. 3.4. Production of 13 C trans-resveratrol by feeding [1-13 C]-l-Phe To produce the 13 C-labeled trans-resveratrol, [1-13 C]-l-Phe (1 mM) was fed into the cultures, elicited by 10 M JA and 500 M SA on day 4, with 200 g L−1 HP2MGL added at the first day. The cells and adsorbents were harvested on days 5–10. The cell growth, 13 C-trans-resveratrol enrichment, and production of anthocyanin and trans-resveratrol are shown in Fig. 4. The results demonstrated that the trans-resveratrol production increased gradually from day 5 to day 10 (from 746.2 mg L−1 to 1144.1 mg L−1 ), and the 13 Cresveratrol enrichment showed an opposite trend (from 35.7% on day 5 to 20.8% on day 10). Similar with natural 13 C abundance [28], the 13 C atom percentage of trans-resveratrol produced in
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Fig. 5.
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C NMR spectra of 13 C-labeled trans-resveratrol. C-4 of trans-resveratrol was 13 C-labeled.
absence of [1-13 C]-l-Phe was 1.09% (laboratory data), when the 13 Ctrans-resveratrol enrichment was only 15.3% (1.09% × 14). From the 9HMBC analysis of 1 H and 13 C, the C-4 of trans-resveratrol was confirmed to be labeled by 13 C (Fig. 5). 4. Discussion Bioproduction of the 13 C-labeled trans-resveratrol in plant cell culture has attracted considerable attention with regard to its potential applications for human health and to better understanding of its absorption and in vivo metabolism in humans and animals [9,18]. There have been several examples of 13 C-labeled trans-resveratrol production [16,18], but low productivity and production from transformation of piceid remain critical problems for industrial production. The typical kinetics of V. vinifera cell culture demonstrated that the cell growth measured as dry cell weight (DCW) exhibited a typical sigmoidal growth curve, reaching a maximum of 17 g L−1 after 10 days of culture. Maximal production of anthocyanins and trans-piceid of about 175 and 440 mg L−1 occurred on days 17 and 14, respectively (Fig. 1). However negligible amount of transresveratrol was produced. The capacity of trans-piceid production in this cell line was more efficient than that used by Krisa et al. [17]. To optimize the production of trans-resveratrol, our laboratory has established an efficient process of combining elicitation by JA and SA with in situ adsorption by HP2MGL in V. vinifera cell culture (Fig. 2). In the absence of HP2MGL, the stilbenes (mainly transpiceid) and anthocyanin were produced, but suppressed by JA and SA. The SA elicitation inhibited the growth and the production of anthocyanin and stilbenes stronger than JA, despite the fact that JA is mostly commonly used for enhancing resveratrol or piceid production [18,22,23,29]. However, the negative effect of JA and SA was overcome by the presence of HP2MGL, which gave enormous transresveratrol production (2666.7 mg L−1 ). When only HP2MGL was added, a decrease in cell growth and improved trans-resveratrol production were observed. This could be explained by the fact that the HP2MGL may work through the mechanism of in situ production removal. Moreover, due to its hydrophobicity, HP2MGL could lead to the adsorption of medium component [21]. It may also be due to the redirection of metabolic flux from anthocyanin pathway to stilbene pathway because significant higher production of trans-resveratrol was achieved with a simultaneous decrease in the production of anthocyanin and trans-piceid. Taking into consider-
ation that stilbenoids and flavonoids are all biosynthesized from cinnamoyl-CoA or -coumaroyl-CoA, which are catalyzed by stilbene synthase (STS) and chalcone synthase (CHS), respectively, our results indicated that in this culture more STSs could be overexpressed, and anthocyanin production was blocked because of the unavailability of substrates [30,31]. In the presence of the adsorbents, only small percentage of stilbenes stored in cells in the form of trans-piceid, and most stilbenes, in the form of trans-resveratrol, were excreted and adsorbed. Similarly, Lijavetzky et al. achieved high trans-resveratrol production (4000 mg L−1 ) in cell suspension culture of V. vinifera cv. Monastrell, with methyl jasmonate and cyclodextrins DIMED. In this work, -cyclodextrins DIMED may have not only acted as elicitors, but also acted to protect transresveratrol in the medium by complex formation [32]. l-Phe feeding results indicated the best feeding time point of day 4 (the beginning of the exponential phase) was independent on the concentrations fed (Fig. 3), similar to the studies of Krisa et al. [17]. Furthermore, higher concentrations of l-Phe feeding (above 1 mM) did not lead to further increase in the trans-resveratrol production (laboratory data). Similarly, Krisa et al. fed 2 mM [1-13 C]-l-Phe repeatedly to increase the enrichment of 13 C but did not achieve significant improvement in the stilbene production. Consequently, the isotopically labeled precursor was fed on day 4 using 1 mM [1-13 C]-l-Phe, for economical reasons. The harvesting time has a significant impact on cell growth, 13 Ctrans-resveratrol enrichment, anthocyanin and trans-resveratrol production after the addition of [1-13 C]-l-Phe on day 4 (Fig. 4). The trans-resveratrol production increased gradually from day 5 to day 10, however, the 13 C-trans-resveratrol enrichment decreased from 35.7% to 20.8%, which was significantly higher than that in absence of [1-13 C]-l-Phe. Owing to the instability of suspension cultures, the trans-resveratrol production at harvest time (day 10) was only 1144.1 mg L−1 in this experiment (Fig. 5), but still comparable with the results of Aumont et al. [16,17]. Results of the 13C NMR indicated that the C-4 of trans-resveratrol was 13 C-labeled (Fig. 5). 5. Conclusions Our results indicated that the rate limiting step was the postbiosynthetic events rather than the biosynthesis in the elicited culture because the stilbenes mainly increased the results through the introduction of HP2MGL adsorbents rather than elicitation
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alone. This optimization strategy was in agreement with Zhang et al., who suggested the importance of simultaneous optimization of the biosynthetic pathways of metabolites and post-biosynthetic steps toward achieving commercial plant cell culture [25]. This biotechnology based on plant cell suspension culture is particularly promising and represents a reliable alternative method under control conditions for production of the non-labeled and isotopically labeled trans-resveratrol, which is usually used for investigation of its absorption and in vivo metabolism in humans and animals. With the help of this technique, we can expect to do scaling-up in a bioreactor to industrially produce large amounts of the non-labeled and 13 C-labeled trans-resveratrol in the future. Acknowledgements The authors are grateful to Dr Song Xue, Dr Xupeng Cao and Professor Xingju Yu for their valuable support. Wei Zhang acknowledges financial support of Chinese National Natural Science Funds (20676130). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bej.2010.12.002. References [1] P. Langcake, R. Pryce, A new class of phytoalexins from grapevine, Cell. Mol. Life Sci. 33 (1977) 151–152. [2] U. Stervbo, O. Vang, C. Bonnesen, A review of the content of the putative chemopreventive phytoalexin resveratrol in red wine, Food Chem. 101 (2007) 449–457. [3] M.J. Takaoka, The phenolic substances of white hellebore (Veratrum grandiflorum Loes. fil.), J. Faculty Sci. Hokkaido Imperial University 3 (1940) 1–16. [4] T. Lanz, G. Schr der, J. Schr der, Differential regulation of genes for resveratrol synthase in cell cultures of Arachis hypogaea L., Planta 181 (1990) 169–175. [5] J. Fliegmann, G. Schr der, S. Schanz, L. Britsch, J. Schr der, Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants, Plant Mol. Biol. 18 (1992) 489–503. [6] A. Kodan, H. Kuroda, F. Sakai, Simultaneous expression of stilbene synthase genes in Japanese red pine (Pinus densiflora) seedlings, J. Wood Sci. 47 (2001) 58–62. [7] B. Aggarwal, A. Bhardwaj, R. Aggarwal, N. Seeram, S. Shishodia, Y. Takada, Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies, Anticancer Res. 24 (2004) 2783. [8] J. Kundu, Y. Surh, Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives, Cancer Lett. 269 (2008) 243–261. [9] D. Donnez, P. Jeandet, C. Clement, E. Courot, Bioproduction of resveratrol and stilbene derivatives by plant cells and microorganisms, Trends Biotechnol. 27 (2009) 706–713. [10] S. Sharma, K. Chopra, S. Kulkarni, Effect of insulin and its combination with resveratrol or curcumin in attenuation of diabetic neuropathic pain: participation of nitric oxide and TNF-alpha, Phytother. Res. 21 (2007) 278–283.
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Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej
Hyper-production of 13 C-labeled trans-resveratrol in Vitis vinifera suspension cell culture by elicitation and in situ adsorption Xiangguo Yue a,b , Wei Zhang a,c,∗ , Maicun Deng b a b c
Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China Graduate School of the Chinese Academy of Sciences, Beijing 100039, China Flinders Centre for Marine Bioprocessing and Bioproducts, and Department of Medical Biotechnology, School of Medicine, Flinders University, Adelaide, SA 5042, Australia
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Article history: Received 6 July 2010 Received in revised form 9 December 2010 Accepted 12 December 2010 Available online 21 December 2010 Keywords: Vitis vinifera 13 C-labeled trans-resveratrol Secondary metabolites Plant cell tissue Culture engineering Metabolic control Adsorption
a b s t r a c t Bioproduction of 13 C-labeled trans-resveratrol in plant cell culture has attracted considerable attention with regard to potential applications for human benefit and to better understanding their absorption and in vivo metabolism in humans and animals. In the present work, two elicitors (SA and JA) and adsorbents (HP2MGL) were introduced into the Vitis vinifera cell suspension culture, and the results indicated that they could work synergistically to improve the production of trans-resveratrol (2666.7 mg L−1 ). Afterward, 1 mM [1-13 C]-l-phenylalanine (Phe) was added to bioproduce 13 C-labeled trans-resveratrol with the 13 C enrichment from 35.7% at day 5 to 20.8% at day 10. Purification of the products by several chromatographic steps was reported, and the 13 C labeling position was verified using 13 C NMR. Our results indicated that, in this case, the rate limiting step of production was the post-biosynthetic events rather than the biosynthesis in elicited culture. Furthermore, our results suggested the importance of simultaneous optimization of biosynthetic pathways of metabolites and their post-biosynthetic steps toward achieving commercial plant cell culture. With the help of this technique, we could expect to do industrial scaling-up in a bioreactor to produce high amounts of the non-labeled and 13 C-labeled trans-resveratrol in the near future. © 2011 Elsevier B.V. All rights reserved.
1. Introduction trans-Resveratrol (3,5,4 -trihydroxystilbene) is a natural compound belonging to the stilbene family. It is produced in response to biotic and abiotic stress, therefore acting like a phytoalexin [1,2]. It was first isolated from white hellebore [3] and has been reported in several plants, such as peanut, lily, mulberries, eucalyptus, spruce and pine [4–6]. However, a Chinese herbal medicinal plant, Polygonum cuspidatum and grapevine are currently the main sources of this compound. Resveratrol has become the focus of a number of studies in medicine and plant physiology, and has emerged as a promising molecule that potentially plays a key role in human health [7–15]. For these reasons, the Vitis vinifera cell suspension culture has been studied as a reliable alternative approach to produce isotopically 13 C-labeled trans-resveratrol for investigation of its absorption and the in vivo metabolism in humans and animals [9,16,17]. Various strategies have been established for the production of 13 C-labeled trans-resveratrol with V. vinifera cell culture. Krisa et al.
∗ Corresponding author at: Marine Bioproducts Engineering Group, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China. E-mail address: [email protected] (W. Zhang). 1369-703X/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2010.12.002
reported that the total piceid reached 770 mol L−1 with the 13 C enrichment rounded to about 66% at day 14, and the piceid can be extracted from cells and transformed to resveratrol by enzymatic hydrolysis [18]. Subsequently, Aumont et al. used a 2 L stirred-tank bioreactor for producing isotopically 13 C-labeled phenolic substances, in which the piceid production reached 360 mg L−1 [16]. However, there is no report of a cell culture system to produce directly 13 C-labeled resveratrol at high productivity. Previously, there have been a number of reports using synthetic adsorbents to remove products in situ [19–21] and using elicitors to improve resveratrol production [18,22,23]. A Spanish research group has achieved a significantly high resveratrol production in grapevine cell culture by combining elicitation and in situ product removal. Cyclodextrins acted as both elicitors and artificial storage in this experiment for product removal [24]. In our previous studies, different types of absorbents and several elicitors were screened and tested, with the most efficient elicitors and absorbents identified for enhancing the trans-resveratrol production (unpublished results). Additionally, 500 M salicylic acid (SA) was observed to increase extracellular phenolics in combination with 10 M jasmonic acid (JA), despite its poor cell growth (unpublished results). As a result, this project aimed to simultaneously use elicitors (SA and JA) and adsorbents for improving the production of 13 C-labeled resveratrol in the V. vinifera cell culture.
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Moreover, this project was also established to test the hypothesis, in which Zhang et al. suggested the importance of simultaneous optimization of biosynthetic pathways of secondary metabolites and their post-biosynthetic steps toward achieving commercial plant cell culture [25]. The production of 13 C labeled trans-resveratrol was achieved by feeding the [1-13 C]-l-Phe, and the 13 C labeling position of 13 C-labeled trans-resveratrol was verified by the 13 C NMR. 2. Materials and methods 2.1. Chemicals [1-13 C]-l-Phe was purchased from Cambridge Isotope Laboratories, Inc. (USA). trans-Resveratrol and trans-piceid, jasmonic acid (JA), salicylic acid (SA) and l-Phe standards were from Sigma (China). HP2MGL was purchased from Rohm and Hass Company (China). All other chemicals used were of analytical grade, and solvents were purchased in HPLC grade. 2.2. V. vinifera cell suspension cultures The cell line used in this study was developed by Cormier et al. [26], originating from a callus established in 1978 from V. vinifera L. cv. Gamay Fréaux var. teinturier berry pulp. This cell line, capable of anthocyanin and trans-piceid accumulation in the dark, was a gift from Dr. Francois Cormier’s group (Quebec, Canada). The cell line has been subcultured in our lab for over 10 years, and subjected to intensive screening of highly pigmented line. The subcultures were performed by inoculating approximately 2.5 g wet cells into 20 mL of the B5 medium, supplemented with 30 g L−1 sucrose, 250 mg L−1 casein hydrolysate, 0.1 mg L−1 ␣-naphthaleneacetic acid (NAA) and 0.2 mg L−1 kinetin. The wet cells were prepared by filtering precultured 7-day-old suspension cells with a 50-m mesh. The subcultures were conducted weekly in the dark, in 500-mL Erlenmeyer flasks, enclosed with an aluminum foil and were maintained on a reciprocating shaker at 100 rpm and 25 ± 1 ◦ C. 2.3. Pre-treatment of the HP2MGL adsorbent The HP2MGL adsorbent, a non-aromatic resin, based on methacrylic ester copolymer, was first rinsed in Milli-Q water using a 1 L measuring cylinder and then transferred into a 1 L glass column (2.5 cm diameter × 40 cm length). The HP2MGL adsorbent was washed with five bed volumes of 100% methanol at a flow rate of two bed volumes per hour. After this, the adsorbent was washed with ten bed volumes of Milli-Q water at a flow rate of five bed volumes per hour. Finally, the HP2MGL adsorbent was vacuum-filtered and ready for application at the beginning of culture. 2.4. Elicitors treatment and precursor feeding Jasmonic acid (JA) and salicylic acid (SA) were dissolved, respectively, in ethanol–water (12:13) and 100% ethanol. l-Phe and [1-13 C]-l-Phe were prepared according to the method of Krisa et al. [18]. Control cultures received the corresponding vehicle solvent at a final concentration that did not exceed 0.2% (ETOH) or 0.5% (DMSO). SA, JA and Phe were filter-sterilized and added into culture at the beginning of the exponential growth phase (day 4). 2.5. Quantification and identification of anthocyanins and stilbenes (trans-resveratrol and trans-piceid) At the time of sampling, the control cultures were harvested by vacuum filtration through filter paper, washed with Milli-Q water and weighed to obtain the fresh cell weight. To separate cells from
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adsorbents in the treatments, 20% sucrose solution was added, and owing to their different densities, the cells can be collected from the top of the medium whereas the adsorbents congregate on the bottom of Erlenmeyer flasks. The anthocyanin existed only in cells and the content was analyzed according to the protocol reported by Zhang et al. [27]. Stilbenes (trans-piceid or trans-resveratrol) were extracted with 20-fold (v/w) MeOH containing 1% HCl and MeOH from cells and adsorbents, respectively. The mixture was incubated overnight at room temperature and then centrifuged for 10 min at 14,000 rpm. The supernatant was obtained for HPLC analysis. The stilbenes (trans-resveratrol and trans-piceid) extracted from cells and adsorbents were identified by co-elution with an authentic standard. Stilbene contents were estimated from a standard curve that was prepared with standards of trans-resveratrol and trans-piceid. 2.6. Purification of 13 C-labeled trans-resveratrol and measurement of 13 C-trans-resveratrol enrichment To purify the trans-resveratrol, the MeOH extracts of adsorbents were evaporated to dryness and the residue was re-dissolved in 5 mL MeOH. The solutions were purified on a reversed-phase C18 column. The fractions containing trans-resveratrol were condensed and purified through a silica gel column followed by elution with methanol and water. Finally, the obtained extracts were purified by semi-preparative HPLC on a SEP-PAK C18 column. The solution containing trans-resveratrol was filtered and vacuum dried to produce a white solid powder, i.e. trans-resveratrol. The above chromatogram operations were all monitored at 286 and 307 nm. The position of 13 C labeling was verified by NMR (Varian INOVA 400 M NMR). The percentage of 13 C enrichment was measured by EA-IRMS (Flash EA 1112 HT, Finnigan MAT Delta V advantage). Samples were injected via an automatic injector into the elemental analyzer and totally converted to CO2 . The CO2 was transferred to the IRMS using helium (120 ml min−1 ). The different isotopomers were separated by a uniform magnetic field and collected in three different collectors at m/z 44, 45 and 46. A known percentage enrichment of [1-13 C]-l-phenylalanine (99%) was regularly analyzed to verify the validity of the method. 13 C atom percentage was transformed to 13 C enrichment percentage with the formula 13 C enrichment (%) = 13 C atom (%) × number of C per molecule [16,18]. 3. Results 3.1. Growth, anthocyanin and stilbenes accumulation of V. vinifera cell suspension culture To understand the growth, anthocyanin and stilbene accumulation in grape cells for determining the optimal period for stilbene production, cultures were analyzed throughout a 21-day period (Fig. 1). Maximal production of anthocyanins and trans-piceid (about 175 and 440 mg L−1 , respectively) occurred on days 17 and 14, respectively. The maximum cell growth of about 17 g DCW L−1 was reached on day 10. In addition, the medium was examined, and either little or no anthocyanin and stilbene was detected (unpublished data). 3.2. Effect of simultaneous elicitation and addition of adsorbents Based on our previous lab results, elicitors (JA 10 M, SA 500 M added on day 4) and HP2MGL (200 g L−1 added at the first day) were introduced to enhance the production of transresveratrol (Fig. 2). The results demonstrated that the cell growth, as well as the production of anthocyanin and stilbenes, were suppressed deleteriously when JA and/or SA was added in the
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Fig. 1. Time course of cell growth, anthocyanin and trans-piceid biosynthesis in Vitis vinifera cell suspension culture. Data represents the mean ± S.D. of three replicates.
Fig. 2. Effect of the addition of JA, SA and HP2MGL on cell growth, anthocyanin, trans-piceid and trans-resveratrol production on the V. vinifera cell suspension culture. JA (10 M) and SA (500 M) were added on day 4. HP2MGL (200 g L−1 ) was added at the first day, and cells and adsorbents were harvested on day 10. Data are the mean ± S.D. of three replicates.
absence of HP2MGL. In the presence of HP2MGL, 1826.7 mg L−1 trans-resveratrol was produced, with a significant decrease in anthocyanin production (from 100 mg L−1 to 42.3 mg L−1 ). However, the cell growth (10.6 DCW L−1 ) was comparable to the control (12.2 DCW L−1 ). When the elicitors and adsorbents were introduced simultaneously, the total production of trans-resveratrol was further improved, with 2666.7 mg L−1 achieved in the presence of adsorbent and both elicitors. The growth and anthocyanin production were suppressed to some extent (4.2 DCW L−1 , and 33.5 mg L−1 , respectively). In all experiments, the trans-resveratrol is secreted into medium and adsorbed in adsorbents, with minor or undetectable level in the cells and medium, while trans-piceid and anthocyanins are accumulated only in the cells. 3.3. Addition of l-Phe in the presence of elicitation and adsorbents The effect of different feeding time points and concentrations of l-Phe on the production of trans-resveratrol was examined (Fig. 3). Three different concentrations (1 mM, 2 mM, and 3 mM) were applied from the beginning to the middle of the exponential phase (days 4–7). The results showed that, regardless of the concentrations, the beginning of exponential phase (day 4) was the best time to maximize the production of trans-resveratrol, similar to the results of Krisa et al. [16,18,23]. Furthermore, addition of
Fig. 3. Effect of feeding time points and concentration of l-Phe on cell growth, anthocyanin and trans-resveratrol production in the V. vinifera cell suspension culture. Different concentrations (1 mM, 2 mM, and 3 mM) l-Phe were added on days 4, 5, 6, 7, respectively. JA (10 M), SA (500 M) and HP2MGL (200 g L−1 ) were added in all the tests except the control. Cells and adsorbents were harvested on day 10. Data are the mean ± S.D. of three replicates.
Fig. 4. Effect of [1-13 C]-l-Phe feeding on cell growth, anthocyanin and transresveratrol production in V. vinifera cell suspension culture, and the 13 C-transresveratrol enrichment. [1-13 C]-l-Phe (1 mM) was added on day 4. JA (10 M), SA (500 M) and HP2MGL (200 g L−1 ) were added in all the tests. Cells and adsorbents were harvested on days 5, 6, 7,8, 9, 10, respectively, Data are the ± S.D. of three replicates
l-Phe (1–10 mM) did not result in a significant increase in production of trans-resveratrol (laboratory data), and l-Phe feeding could slightly suppress the anthocyanin production. The optimal production conditions of 13 C-labeled trans-resveratrol were established as feeding 1 mM [1-13 C]-l-Phe on day 4, when 10 M JA and 500 M SA were added on day 4, with the presence of 200 g L−1 HP2MGL from the first day. 3.4. Production of 13 C trans-resveratrol by feeding [1-13 C]-l-Phe To produce the 13 C-labeled trans-resveratrol, [1-13 C]-l-Phe (1 mM) was fed into the cultures, elicited by 10 M JA and 500 M SA on day 4, with 200 g L−1 HP2MGL added at the first day. The cells and adsorbents were harvested on days 5–10. The cell growth, 13 C-trans-resveratrol enrichment, and production of anthocyanin and trans-resveratrol are shown in Fig. 4. The results demonstrated that the trans-resveratrol production increased gradually from day 5 to day 10 (from 746.2 mg L−1 to 1144.1 mg L−1 ), and the 13 Cresveratrol enrichment showed an opposite trend (from 35.7% on day 5 to 20.8% on day 10). Similar with natural 13 C abundance [28], the 13 C atom percentage of trans-resveratrol produced in
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Fig. 5.
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C NMR spectra of 13 C-labeled trans-resveratrol. C-4 of trans-resveratrol was 13 C-labeled.
absence of [1-13 C]-l-Phe was 1.09% (laboratory data), when the 13 Ctrans-resveratrol enrichment was only 15.3% (1.09% × 14). From the 9HMBC analysis of 1 H and 13 C, the C-4 of trans-resveratrol was confirmed to be labeled by 13 C (Fig. 5). 4. Discussion Bioproduction of the 13 C-labeled trans-resveratrol in plant cell culture has attracted considerable attention with regard to its potential applications for human health and to better understanding of its absorption and in vivo metabolism in humans and animals [9,18]. There have been several examples of 13 C-labeled trans-resveratrol production [16,18], but low productivity and production from transformation of piceid remain critical problems for industrial production. The typical kinetics of V. vinifera cell culture demonstrated that the cell growth measured as dry cell weight (DCW) exhibited a typical sigmoidal growth curve, reaching a maximum of 17 g L−1 after 10 days of culture. Maximal production of anthocyanins and trans-piceid of about 175 and 440 mg L−1 occurred on days 17 and 14, respectively (Fig. 1). However negligible amount of transresveratrol was produced. The capacity of trans-piceid production in this cell line was more efficient than that used by Krisa et al. [17]. To optimize the production of trans-resveratrol, our laboratory has established an efficient process of combining elicitation by JA and SA with in situ adsorption by HP2MGL in V. vinifera cell culture (Fig. 2). In the absence of HP2MGL, the stilbenes (mainly transpiceid) and anthocyanin were produced, but suppressed by JA and SA. The SA elicitation inhibited the growth and the production of anthocyanin and stilbenes stronger than JA, despite the fact that JA is mostly commonly used for enhancing resveratrol or piceid production [18,22,23,29]. However, the negative effect of JA and SA was overcome by the presence of HP2MGL, which gave enormous transresveratrol production (2666.7 mg L−1 ). When only HP2MGL was added, a decrease in cell growth and improved trans-resveratrol production were observed. This could be explained by the fact that the HP2MGL may work through the mechanism of in situ production removal. Moreover, due to its hydrophobicity, HP2MGL could lead to the adsorption of medium component [21]. It may also be due to the redirection of metabolic flux from anthocyanin pathway to stilbene pathway because significant higher production of trans-resveratrol was achieved with a simultaneous decrease in the production of anthocyanin and trans-piceid. Taking into consider-
ation that stilbenoids and flavonoids are all biosynthesized from cinnamoyl-CoA or -coumaroyl-CoA, which are catalyzed by stilbene synthase (STS) and chalcone synthase (CHS), respectively, our results indicated that in this culture more STSs could be overexpressed, and anthocyanin production was blocked because of the unavailability of substrates [30,31]. In the presence of the adsorbents, only small percentage of stilbenes stored in cells in the form of trans-piceid, and most stilbenes, in the form of trans-resveratrol, were excreted and adsorbed. Similarly, Lijavetzky et al. achieved high trans-resveratrol production (4000 mg L−1 ) in cell suspension culture of V. vinifera cv. Monastrell, with methyl jasmonate and cyclodextrins DIMED. In this work, -cyclodextrins DIMED may have not only acted as elicitors, but also acted to protect transresveratrol in the medium by complex formation [32]. l-Phe feeding results indicated the best feeding time point of day 4 (the beginning of the exponential phase) was independent on the concentrations fed (Fig. 3), similar to the studies of Krisa et al. [17]. Furthermore, higher concentrations of l-Phe feeding (above 1 mM) did not lead to further increase in the trans-resveratrol production (laboratory data). Similarly, Krisa et al. fed 2 mM [1-13 C]-l-Phe repeatedly to increase the enrichment of 13 C but did not achieve significant improvement in the stilbene production. Consequently, the isotopically labeled precursor was fed on day 4 using 1 mM [1-13 C]-l-Phe, for economical reasons. The harvesting time has a significant impact on cell growth, 13 Ctrans-resveratrol enrichment, anthocyanin and trans-resveratrol production after the addition of [1-13 C]-l-Phe on day 4 (Fig. 4). The trans-resveratrol production increased gradually from day 5 to day 10, however, the 13 C-trans-resveratrol enrichment decreased from 35.7% to 20.8%, which was significantly higher than that in absence of [1-13 C]-l-Phe. Owing to the instability of suspension cultures, the trans-resveratrol production at harvest time (day 10) was only 1144.1 mg L−1 in this experiment (Fig. 5), but still comparable with the results of Aumont et al. [16,17]. Results of the 13C NMR indicated that the C-4 of trans-resveratrol was 13 C-labeled (Fig. 5). 5. Conclusions Our results indicated that the rate limiting step was the postbiosynthetic events rather than the biosynthesis in the elicited culture because the stilbenes mainly increased the results through the introduction of HP2MGL adsorbents rather than elicitation
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alone. This optimization strategy was in agreement with Zhang et al., who suggested the importance of simultaneous optimization of the biosynthetic pathways of metabolites and post-biosynthetic steps toward achieving commercial plant cell culture [25]. This biotechnology based on plant cell suspension culture is particularly promising and represents a reliable alternative method under control conditions for production of the non-labeled and isotopically labeled trans-resveratrol, which is usually used for investigation of its absorption and in vivo metabolism in humans and animals. With the help of this technique, we can expect to do scaling-up in a bioreactor to industrially produce large amounts of the non-labeled and 13 C-labeled trans-resveratrol in the future. Acknowledgements The authors are grateful to Dr Song Xue, Dr Xupeng Cao and Professor Xingju Yu for their valuable support. Wei Zhang acknowledges financial support of Chinese National Natural Science Funds (20676130). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bej.2010.12.002. References [1] P. Langcake, R. Pryce, A new class of phytoalexins from grapevine, Cell. Mol. Life Sci. 33 (1977) 151–152. [2] U. Stervbo, O. Vang, C. Bonnesen, A review of the content of the putative chemopreventive phytoalexin resveratrol in red wine, Food Chem. 101 (2007) 449–457. [3] M.J. Takaoka, The phenolic substances of white hellebore (Veratrum grandiflorum Loes. fil.), J. Faculty Sci. Hokkaido Imperial University 3 (1940) 1–16. [4] T. Lanz, G. Schr der, J. Schr der, Differential regulation of genes for resveratrol synthase in cell cultures of Arachis hypogaea L., Planta 181 (1990) 169–175. [5] J. Fliegmann, G. Schr der, S. Schanz, L. Britsch, J. Schr der, Molecular analysis of chalcone and dihydropinosylvin synthase from Scots pine (Pinus sylvestris), and differential regulation of these and related enzyme activities in stressed plants, Plant Mol. Biol. 18 (1992) 489–503. [6] A. Kodan, H. Kuroda, F. Sakai, Simultaneous expression of stilbene synthase genes in Japanese red pine (Pinus densiflora) seedlings, J. Wood Sci. 47 (2001) 58–62. [7] B. Aggarwal, A. Bhardwaj, R. Aggarwal, N. Seeram, S. Shishodia, Y. Takada, Role of resveratrol in prevention and therapy of cancer: preclinical and clinical studies, Anticancer Res. 24 (2004) 2783. [8] J. Kundu, Y. Surh, Cancer chemopreventive and therapeutic potential of resveratrol: mechanistic perspectives, Cancer Lett. 269 (2008) 243–261. [9] D. Donnez, P. Jeandet, C. Clement, E. Courot, Bioproduction of resveratrol and stilbene derivatives by plant cells and microorganisms, Trends Biotechnol. 27 (2009) 706–713. [10] S. Sharma, K. Chopra, S. Kulkarni, Effect of insulin and its combination with resveratrol or curcumin in attenuation of diabetic neuropathic pain: participation of nitric oxide and TNF-alpha, Phytother. Res. 21 (2007) 278–283.
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