Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots

Plant Cell, Tissue and Organ Culture - Tập 119 - Trang 65-77 - 2014
Mariacarmela Vaccaro1, Nicola Malafronte1, Mariaevelina Alfieri1, Nunziatina De Tommasi1, Antonietta Leone1
1Department of Pharmacy, University of Salerno, Fisciano, Italy

Tóm tắt

Diterpenoids are important compounds for plant survival and have beneficial properties for humans. Bioactive abietanic diterpenes are synthesized in roots of Salvia sclarea (e.g. aethiopinone, 1-oxoaethiopinone, salvipisone, and ferruginol), but at a very low level (about 1 % of root dry weight). To enhance the biosynthesis of this interesting class of compounds, heterologous AtDXS (d-xylulose 5-phosphate synthase) or AtDXR (1-deoxy-d-xylulose 5 phosphate reductoisomerase) genes, encoding the up-stream enzymes of the plastidial 2-C-methyl-D-erythritol 4-phosphate (MEP)-dependent terpenoid pathway, were ectopically expressed in S. sclarea hairy roots. Quantitative targeted metabolic analysis (HPLC–DAD) revealed that three independent root lines, expressing different levels of DXS or DXR transcripts and proteins, synthesized a significant higher content of abietanic diterpenes, compared to the control hairy root line transformed with the empty vector. The increase was gene-dependent, since the overexpression of the AtDXR triggered a 4.4-fold increase in aethiopinone, an abietane quinone-type tricyclic diterpene. In addition, aethiopinone was proved to be cytotoxic to different solid tumor cell lines, with the highest effect on human melanoma A375 cell line (IC50 11.4 µM). Overall these results show that it is possible to boost the metabolic flow towards the synthesis of abietanic diterpenes in S. sclarea hairy roots by overexpressing genes involved in the first steps of the MEP-pathway and provide new insights for the large-scale production of this class of compounds, with potential application in cancer treatment.

Tài liệu tham khảo

Alasbahi RH, Melzig MF (2010) Plectranthus barbatus: a review of phytochemistry, ethnobotanical uses and pharmacology—Part 2. Planta Med 76:753–765

Bede JC, Musser RO, Felton GW, Korth KL (2006) Caterpillar herbivory and salivary enzymes decrease transcript levels of Medicago truncatula genes encoding early enzymes in terpenoid biosynthesis. Plant Mol Biol 60:519–531

Caniard A, Zerbe P, Legrand S, Cohade A, Valot N, Magnard JL, Bohlmann J, Legendre L (2012) Discovery and functional characterization of two diterpene synthase for sclareol biosynthesis in Salvia sclarea (L.) and their relevance for perfume manufacture. BMC Plant Biol 12:119–132

Carretero-Paulet L, Ahumada I, Cunillera N, Rodríguez-Concepción M, Ferrer A, Boronat A, Campos N (2002) Expression and molecular analysis of the Arabidopsis DXR gene enconding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Plant Physiol 129:1581–1591

Carretero-Paulet L, Cairó A, Botella-Pavía P, Besumbes O, Campos N, Boronat A, Rodríguez-Concepción M (2006) Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxylulose 5-phosphate reductoisomerase. Plant Mol Biol 62:683–695

Chappell J (1995) Biochemistry and molecular biology of the isoprenoid pathway in plants. Annu Rev Plant Mol Biol 46:521–547

Cordoba E, Salmi M, León P (2009) Unravelling the regulatory mechanisms that modulate the MEP pathway in higher plants. J Exp Bot 60:2933–2943

Dat NT, Jin X, Lee JH, Lee D, Hong YS, Lee K, Kim YH, Lee JJ (2007) Abietane diterpenes from Salvia miltiorrhiza inhibit the activation of hypoxia-inducible factor-1. J Nat Prod 70:1093–1097

Doyle JJ, Doyle JL (1990) A rapid total DNA preparation procedure for fresh plant tissue. Focus 12:13–15

Dudareva N, Andersson S, Orlova I, Gatto N, Reichelt M, Rhodes D, Boland W, Gerchenzon J (2005) The nonmevalonate pathway supportos both monoterpene and sesquiterpenes formation in snapdragon flowers. Proc Natl Acad Sci USA 102:933–938

Dudareva N, Klempien A, Muhlemann JK, Kaplan I (2013) Biosynthesis, function and metabolic engineering of plant volatile organic compounds. New Phytol 198:16–32

Enfissi EM, Fraser PD, Lois LM, Boronat A, Schuch W, Bramley PM (2005) Metabolic engineering of the mevalonate and non mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato. Plant Biotech J 3:17–27

Estévez JM, Cantero A, Reindl A, Reichler S, León P (2001) 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J Biol Chem 276:22901–22909

Fronza M, Lamy E, Günther S, Heinzmann B, Laufer S, Merfort I (2012) Abietane diterpenes induce cytotoxic effects in human pancreatic cancer cell line MIA PaCa-2 through different modes of action. Phytochemistry 78:107–119

Georgiev M, Agostini E, Ludwig-Muller J, Xu J (2012) Genetically transformed roots: from plant disease to biotechnological resource. Trends Biotech 30:528–537

Haas JH, Moore LW, Ream W, Manulis S (1995) Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Appl Environ Microbiol 61:2879–2884

Hans J, Hause B, Strack D, Walter MH (2004) Cloning, characterization, and immunolocalization of a mycorrhiza-inducible 1-deoxy-D-xylulose 5-phosphate reductoisomerase in arbuscule-containing cells of maize. Plant Physiol 134:614–624

Hasunuma T, Takeno S, Hayashi S, Sendai M, Bamba T, Yoshimura S, Tomizawa K, Fukusaki E, Miyake C (2008) Overexpression of 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production. J Biosci Bioeng 105:518–526

Hasunuma T, Kondo A, Miyake C (2010) Metabolic engineering by plastid transformation as a strategy to modulate isoprenoid yield in plants. Methods Mol Biol 643:213–227

Hsieh MH, Goodman HM (2005) The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis. Plant Physiol 138:641–653

Jennewein S, Croteau R (2001) Taxol: biosynthesis, molecular genetics, and biotechnological applications. Appl Microbiol Biotechnol 57:13–19

Kai G, Xu H, Zhou C, Liao P, Xiao J, Luo X, You L, Zhang L (2011) Metabolic engineering tanshinone biosynthetic pathway in Salvia miltiorrhiza hairy root cultures. Metab Engin 13:319–327

Kuźma Ł, Skrzypek Z, Wysokińska H (2006) Diterpenoids and triterpenoids in hairy roots of Salvia sclarea. Plant Cell Tissue Org Cult 84:171–179

Kuźma L, Rozalsk M, Walencka E, Rozalska B, Wysokinska H (2007) Antimicrobial activity of diterpenoids from hairy roots of Salvia sclarea L: salvipisone as a potential antibiofilm agent active against antibiotic resistant staphylococci. Phytomedicine 14:31–35

Lange BM, Wildung MR, McCaskill D, Croteau R (1998) A family of transketolases that directs isoprenoid biosynthesis via a mevalonate-independent pathway. Proc Nat Acad Sci USA 95:2100–2104

Lange BM, Rujan T, Martin W, Croteau R (2000) Isoprenoid biosynthesis: the evolution of two ancient and distinct pathways across genomes. Proc Natl Acad Sci USA 97:13172–13177

Li L, Shukla S, Lee A, Garfield SH, Maloney DJ, Ambudkar SV, Yuspa SH (2010) The skin cancer chemotherapeutic agent ingenol-3-angelate (PEP005) is a substrate for the epidermal multidrug transporter (ABCB1)and targets tumor vasculature. Cancer Res 70:4509–4519

Lois LM, Campos N, Putra SR, Danielsen K, Rohmer M, Boronat A (1998) Cloning and characterization of a gene from Escherichia coli encoding a tranketolase-like enzyme that catalyzes the synthesis of D-1-deoxyxylulose 5 –phosphate, a common precursor for isoprenoid, thiamin, and pyridoxol biosynthesis. Proc Natl Acad Sci USA 95:2105–2110

Lu Q, Zhang P, Zhang X, Chen J (2009) Experimental study of the anti-cancer mechanism of tanshinone IIA against human breast cancer. J Int Mol Med 24:773–780

Mahaira LG, Tsimplouli C, Sakellaridis N, Alevizopoulos K, Demetzos C, Han Z, Pantazis P, Dimas K (2011) The labdane diterpene sclareol (labd-14-ene-8, 13-diol) induces apoptosis in human tumor cell lines and suppression of tumor growth in vivo via a p53-independent mechanism of action. Eur J Pharmacol 666:173–182

Mahmoud SS, Croteau RB (2001) Metabolic engineering of essential oil yield and composition in mint by altering expression of deoxy-xylulose phosphate reductoisomerase and menthofuran synthase. Proc Nat Acad Sci USA 98:8915–8920

Mayrhofer S, Teuber M, Zimmer I, Louis S, Fischbach RJ, Schnitzler JP (2005) Diurnal and seasonal variation of isoprene biosynthesis-related genes in grey poplar leaves. Plant Physiol 139:474–484

Miñoz-Bertomeu J, Arrillaga I, Ros R, Segura J (2006) Up-regulation of 1-deoxy-D-xylulose-5-phosphate synthase enhances production of essential oils in transgenic spike lavender. Plant Physiol 142:890–900

Mnonopi N, Levendal RA, Mzilikazi N, Frost CL (2012) Marrubiin, a constituent of Leonotis leonurus, alleviates diabetic symptoms. Phytomedicine 19:488–493

Morris WL, Ducreux LJ, Hedden P, Millam S, Taylor MA (2006) Overexpression of a bacterial 1-deoxy-D-xylulose 5-phosphate synthase gene in potato tubers perturbs the isoprenoid metabolic network: implications for the control of the tuber life cycle. J Exp Bot 57:3007–3018

Moses T, Pollier J, Thevelein JM, Goossens A (2013) Bioengineering of plant (tri)terpenoids: from metabolic engineering of plants to synthetic biology in vivo and in vitro. New Phytol 200:27–43

Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63

Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Plant Physiol 15:473–497

Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocite apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139:271–279

Noori S, Hassan ZM, Mohammad M, Habibi Z, Sohrabi N, Bavanolhagh S (2010) Sclareol modulates the Treg intra-tumoral infiltrated cell and inhibits tumor growth in vivo. Cell Immunol 263:148–153

Opipari AWJr, Hu HM, Yabkowitz R, Dixit VM (1992) The A20 zinc-finger protein protects cells from turmor necrosis factor cytotoxicity. J Biol Chem 297:12424–12427

Pan TL, Hung YC, Wang PW, Chen ST, Hsu TK, Sintupisut N, Cheng CS, Lyu PC (2010) Functional proteomic and structural insights into molecular targets related to the growth inhibitory effect of tanshinone IIA on HeLa cells. Proteomics 10:914–929

Peebles AM, Sander G, Hughes E, Peacok R, Shanks J, San K (2011) The expression of 1-deoxy-D-xylulose synthase and geraniol-10-hydroxylase or anthranilate synthase increases terpenoid indole alkaloid accumulation in Catharanthus roseus hairy roots. Metab Eng 13:234–240

Phillips M, León P, Boronat A, Rodríguez-Concepción M (2008) The plastidial MEP pathway unified nomenclature and resources. Trend Plant Sci 13:619–623

Pulido P, Toledo-Ortiz G, Phillips MA, Wright LP, Rodríguez-Concepción M (2013) Arabidopsis j-protein j20 delivers the first enzyme of the plastidial isoprenoid pathway to protein quality control. Plant Cell 25:4183–4194

Rigel DS, Carucci JA (2000) Malignant melanoma: prevention, early detection, and treatment in the 21st century. J CA Cancer Clin 50:215–236

Rodríguez-Concepción M, Boronat A (2002) Elucidation of the methylerythritol phosphate pathway for isoprenoid biosynthesis in bacteria and plastids. A metabolic milestone achieved through genomics. Plant Physiol 130:1079–1089

Rodríguez-Concepción M, Ahumada I, Diez-Juez E, Sauret-Gueto S, Lois LM, Gallego F, Carretero Paulet L, Campos N, Boronat A (2001) 1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoids biosynthesis during tomato fruit ripening. Plant J 27:213–222

Rohmer M, Seeman M, Horbach S, Bringer-Meyer S, Sahm H (1996) Glyceraldehyde 3-phosphate and pyruvate as precursors of isoprenic units in an alternative non-mevalonate pathway for terpenoid biosynthesis. J Am Chem Soc 118:2564–2566

Rózalski M, Kuźma L, Krajewska U, Wysokinska H (2006) Cytotoxic and proapoptotic activity of diterpenoids from in vitro cultivated Salvia sclarea roots. Studies on the leukemia cell lines. Z Naturforsch 61:483–488

Schardl CL, Byrd AD, Bezion G, Altschuler MA, Hildebrand DF, Hunt AG (1987) Design and construction of a versatile system for the expression of foreign genes in plants. Gene 61:1–11

Talano M, Oller A, González P, Agostini E (2012) Hairy roots their multiple applications and recents patents. Recent Plant Biotech 6:115–133

Topcu G, Goren AC (2007) Biological activity of diterpenoids isolated from Anatolian laminaceae plants. Rec Nat Prod 1:1–16

Ulubelen A, Eriş C, Sönmez U, Kartal M, Kurucu S, Bozok-Johansson C (1994) Terpenoids from Salvia sclarea. Phytochemistry 36:971–974

Ulubelen A, Sonmez U, Topcu G (1997) Diterpenoids from the roots of Salvia sclarea. Phytochemistry 344:1297–1299

Veau B, Courtois A, Oudin J, Chenieux C, Rideau M, Clastre M (2000) Cloning and expression of cDNAs enconding two enzymes of the MEP pathway in Catharanthus roseus. Biochem Biophys Acta Gene Struct Exp 1517:159–163

Walter M, Fester T, Strack D (2000) Arbuscular mycorrhizal fungi induce the non-mevalonate methylerythritol phosphate pathway of isoprenoid biosynthesis correlated with the accumulation of the yellow pigment and other apocarotenoids. Plant J 21:571–578

Weigel D, Glazebrook J (2006) Transformation of Agrobacterium using the freeze-thaw method. CSH Protoc 7:110–115

Wu S, Shalk M, Clark A, Miles RB, Coates R, Chappell J (2006) Redirection of cytosolic or plastidic isoprenoid precursors elevates terpene production in plants. Nat Biotech 24:1141–1147

Xiang L, Zeng L, Yuan Y, Chen M, Wang F, Liu X, Zeng L, Lan X, Liao Z (2012) Enanchement of artemisinin biosynthesis by overexpressing dxr, cyp71av1 and cpr in the plants of Artemisia annua L. Plant Omics J 5:503–507

Yang L, Ding G, Lin H, Cheng H, Kong Y, Wei Y, Fang X, Liu R, Wang L, Chen X, Yang C (2013) Transcriptome analysis of medicinal plant Salvia miltiorrhiza and identification of genes related to tanshinone biosynthesis. PLoS ONE 8:e80464

Yoon V, Nodwell JR (2014) Activating secondary metabolism with stress and chemicals. J Ind Microbiol Biotechnol 41:415–424

Zerbe P, Hamberger B, Yuen MM, Chiang A, Sandhu HK, Madilao LL, Nguyen A, Hamberger B, Bach SS, Bohlmann J (2013) Gene discovery of modular diterpene metabolism in non model systems. Plant Physiol 162:1073–1091

Zhou L, Chan WK, Xu N, Xiao K, Luo H, Luo KQ, Chang DC (2008) Tanshinone IIA, an isolated compound from Salvia miltiorrhiza Bunge, induces apoptosis in HeLa cells through mitotic arrest. Life Sci 83:394–403

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Plant Cell, Tissue and Organ Culture

Tập 119

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