Planta Med 2019; 85(11/12): 973-980
DOI: 10.1055/a-0895-5878
Biological and Pharmacological Activities
Original Papers
Georg Thieme Verlag KG Stuttgart · New York

High Yield of Bioactive Abietane Diterpenes in Salvia sclarea Hairy Roots by Overexpressing Cyanobacterial DXS or DXR Genes

Mariacarmela Vaccaro
Department of Pharmacy, DIFARMA, University of Salerno, Fisciano, Italy
,
Vivian Ocampo Bernal
Department of Pharmacy, DIFARMA, University of Salerno, Fisciano, Italy
,
Nicola Malafronte
Department of Pharmacy, DIFARMA, University of Salerno, Fisciano, Italy
,
Nunziatina De Tommasi
Department of Pharmacy, DIFARMA, University of Salerno, Fisciano, Italy
,
Antonietta Leone
Department of Pharmacy, DIFARMA, University of Salerno, Fisciano, Italy
› Institutsangaben
Weitere Informationen

Publikationsverlauf

received 21. Januar 2019
revised 29. März 2019

accepted 09. April 2019

Publikationsdatum:
26. April 2019 (online)

Abstract

Abietane diterpenoids, containing a quinone moiety, are synthesized in the roots of several Salvia species. Promising cytotoxicity and antiproliferative activities have been reported for these compounds in various cell and animal models. We have recently shown that aethiopinone, an o-naphto-quinone diterpene, produced in the roots of different Salvia species, is selectively cytotoxic against the A375 melanoma cell line. To enhance the synthesis of this abietane diterpenoid, we have engineered the plastidial 2-C-methyl-D-erythritol 4-phosphate-derived isoprenoid pathway in Salvia sclarea hairy roots by ectopic expression and plastid targeting of cyanobacterial genes encoding the 1-deoxy-D-xylulose 5-phosphate synthase or 1-deoxy-D-xylulose-5-phosphate reductoisomerase gene, the first two enzymatic steps of the plastidial MEP pathway, from which plant diterpenes primarily derive. Plastid-targeted expression of 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase proteins significantly enhanced the yield of aethiopinone by a 3-fold and about 6-fold increase, respectively. The accumulation of other abietane-type diterpenes (ferruginol, salvipisone, and carnosic acid), with interesting antiproliferative activity, was also increased. Compared to our previous data obtained by overexpressing the plant orthologous 1-deoxy-D-xylulose 5-phosphate synthase and 1-deoxy-D-xylulose-5-phosphate reductoisomerase genes in S. sclarea hairy roots, the results presented here confirm that the bacterial 1-deoxy-D-xylulose-5-phosphate reductoisomerase enzyme plays a major role than the DXS enzyme in the biosynthetic pathway of this class of compounds and that its ectopic expression does not conflict with active hairy root growth, resulting in a balanced trade-off between the transgenic hairy root final biomass and the increased content of o-naphto-quinone diterpenes, with interesting biological activities.

Supporting Information

 
  • References

  • 1 Lu JJ, Bao JL, Wu GS, Xu WS, Huang MQ, Chen XP, Wang YT. Quinones derived from plant secondary metabolites as anti-cancer agents. Anticancer Agents Med Chem 2013; 13: 456-463
  • 2 Cragg GM, Newman DJ. Natural products: a continuing source of novel drug leads. Biochim Biophys Acta 2013; 1830: 3670-3695
  • 3 Zhang Y, Jiang P, Ye M, Kim SH, Jiang C, Lü J. Tanshinones: sources, pharmacokinetics and anti-cancer activities. Int J Mol Sci 2012; 13: 13621-13666
  • 4 Bispo de Jesus M, Zambuzzi WF, Ruela de Sousa RR, Areche C, Santos de Souza AC, Aoyama H, Schmeda-Hirschmann G, Rodríguez JA, Monteiro de Souza Brito AR, Peppelenbosch MP, den Hertog J, de Paula E, Ferreira CV. Ferruginol suppresses survival signaling pathways in androgen-independent human prostate cancer cells. Biochimie 2008; 90: 843-854
  • 5 Fronza M, Murillo R, Slusarczyk S, Adams M, Hamburger M, Heinzmann B, Laufer S, Merfort I. In vitro cytotoxic activity of abietane diterpenes from Peltodon longipes as well as Salvia miltiorrhiza and Salvia sahendica . Bioorg Med Chem 2011; 19: 4876-4881
  • 6 Ho ST, Tung YT, Kuo YH, Lin CC, Wu JH. Ferruginol inhibits non-small cell lung cancer growth by inducing caspase-associated apoptosis. Integr Cancer Ther 2015; 14: 86-97
  • 7 Rózalski M, Kuźma L, Krajewska U, Wysokinska H. Cytotoxic and proapoptotic activity of diterpenoids from in vitro cultivated Salvia sclarea roots. Studies on the leukemia cell lines. Z Naturforsch 2006; 61: 483-488
  • 8 Vaccaro MC, Malafronte N, Alfieri M, De Tommasi N, Leone A. Enhanced biosynthesis of bioactive abietane diterpenes by overexpressing AtDXS or AtDXR genes in Salvia sclarea hairy roots. Plant Tiss Org Cult 2014; 119: 65-77
  • 9 Marchev A, Haas C, Schulz S, Georgiev V, Steingroewer J, Bley T, Pavlov A. Sage in vitro cultures: a promising tool for the production of bioactive terpenes and phenolic substances. Biotechnol Lett 2014; 36: 211-221
  • 10 Atanasov AG, Waltenberger B, Pferschy-Wenzig EM, Linder T, Wawrosch C, Uhrin P, Temml V, Wang L, Schwaiger S, Heiss EH, Rollinger JM, Schuster D, Breuss JM, Bochkov V, Mihovilovic MD, Kopp B, Bauer R, Dirsch VM, Stuppner H. Discovery and resupply of pharmacologically active plant-derived natural products: A review. Biotechnol Adv 2015; 33: 1582-1614
  • 11 Tatsis EC, OʼConnor SE. New developments in engineering plant metabolic pathways. Curr Opin Biotechnol 2016; 42: 126-132
  • 12 Boutanaev AM, Moses T, Zi J, Nelson DR, Mugford ST, Peters RJ, Osbourn A. Investigation of terpene diversification across multiple sequenced plant genomes. Proc Natl Acad Sci U S A 2015; 112: E81-E88
  • 13 Lois LM, Rodríguez-Concepción M, Gallego F, Campos N, Boronat A. Carotenoid biosynthesis during tomato fruit development: regulatory role of 1-deoxy-D-xylulose 5-phosphate synthase. Plant J 2000; 22: 503-513
  • 14 Estévez JM, Cantero A, Reindl A, Reichler S, León P. 1-Deoxy-D-xylulose-5-phosphate synthase, a limiting enzyme for plastidic isoprenoid biosynthesis in plants. J Biol Chem 2001; 276: 22901-22909
  • 15 Mahmoud SS, Croteau RB. 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 U S A 2001; 98: 8915-8920
  • 16 Rodríguez-Concepción M, Ahumada I, Diez-Juez E, Sauret-Gueto S, Lois LM, Gallego F, Carretero Paulet L, Campos N, Boronat A. 1-Deoxy-D-xylulose 5-phosphate reductoisomerase and plastid isoprenoid biosynthesis during tomato fruit ripening. Plant J 2001; 27: 213-222
  • 17 Enfissi EM, Fraser PD, Lois LM, Boronat A, Schuch W, Bramley PM. Metabolic engineering of the mevalonate and non-mevalonate isopentenyl diphosphate-forming pathways for the production of health-promoting isoprenoids in tomato. Plant Biotech J 2005; 3: 17-27
  • 18 Carretero-Paulet L, Cairó A, Botella-Pavía P, Besumbes O, Campos N, Boronat A, Rodríguez-Concepción M. Enhanced flux through the methylerythritol 4-phosphate pathway in Arabidopsis plants overexpressing deoxylulose 5-phosphate reductoisomerase. Plant Mol Biol 2006; 62: 683-695
  • 19 Muñoz-Bertomeu J, Arrillaga I, Ros R, Segura J. Up-regulation of 1-deoxy-D-xylulose-5-phosphate synthase enhances production of essential oils in transgenic spike lavender. Plant Physiol 2006; 142: 890-900
  • 20 Hasunuma T, Takeno S, Hayashi S, Sendai M, Bamba T, Yoshimura S, Tomizawa K, Fukusaki E, Miyake C. Overexpression of 1-Deoxy-D-xylulose-5-phosphate reductoisomerase gene in chloroplast contributes to increment of isoprenoid production. J Biosci Bioeng 2008; 105: 518-526
  • 21 Vaccaro MC, Alfieri M, Malafronte N, De Tommasi N, Leone A. Increasing the synthesis of bioactive abietane diterpenes in Salvia sclarea hairy roots by elicited transcriptional reprogramming. Plant Cell Rep 2017; 36: 375-386
  • 22 Alfieri M, Vaccaro M, Cappetta E, Ambrosone A, De Tommasi N, Leone A. Coactivation of MEP-biosynthetic genes and accumulation of abietane diterpenes in Salvia sclarea by heterologous expression of WRKY and MYC2 transcription factors. Sci Rep 2018; 8: 11009
  • 23 Baulcombe D. RNA silencing in plants. Nature 2004; 431: 356-363
  • 24 Raven JA, Allen JF. Genomics and chloroplast evolution: what did cyanobacteria do for plants?. Genome Biol 2003; 4: 209-213
  • 25 Veau B, Courtois A, Oudin J, Chenieux C, Rideau M, Clastre M. Cloning and expression of cDNAs encoding two enzymes of the MEP pathway in Catharanthus roseus . Biochem Biophys Acta Gen St Exp 2000; 1517: 159-163
  • 26 Hans J, Hause B, Strack D, Walter MH. Cloning, characterization, and immunolocalization of a mycorrhiza-inducible 1-deoxy-D-xylulose 5-phosphate reductoisomerase in arbuscule-containing cells of maize. Plant Physiol 2004; 134: 614-624
  • 27 Hsieh MH, Goodman HM. The Arabidopsis IspH homolog is involved in the plastid nonmevalonate pathway of isoprenoid biosynthesis. Plant Physiol 2005; 138: 641-653
  • 28 Mayrhofer S, Teuber M, Zimmer I, Louis S, Fischbach RJ, Schnitzler JP. Diurnal and seasonal variation of isoprene biosynthesis-related genes in grey poplar leaves. Plant Physiol 2005; 139: 474-484
  • 29 Bede JC, Musser RO, Felton GW, Korth KL. Caterpillar herbivory and salivary enzymes decrease transcript levels of Medicago truncatula genes encoding early enzymes in terpenoid biosynthesis. Plant Mol Bio 2006; 60: 519-531
  • 30 Carretero-Paulet L, Ahumada I, Cunillera N, Rodríguez-Concepción M, Ferrer A, Boronat A, Campos N. Expression and molecular analysis of the Arabidopsis DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase, the first committed enzyme of the 2-C-methyl-D-erythritol 4-phosphate pathway. Plant Physiol 2002; 129: 1581-1591
  • 31 Ershov YV, Gantt RR, Cunningham jr. FX, Gantt E. Isoprenoid biosynthesis in Synechocystis sp. strain PCC6803 is stimulated by compounds of the pentose phosphate cycle but not by pyruvate or deoxyxylulose-5-phosphate. J Bacteriol 2002; 184: 5045-5051
  • 32 Battilana J, Emanuelli F, Gambino G, Gribaudo I, Gasperi F, Boss PK, Grando MS. Functional effect of grapevine 1-deoxy-D-xylulose 5-phosphate synthase substitution K284N on Muscat flavour formation. J Exp Bot 2011; 62: 5497-5508
  • 33 Seetang-Nun Y, Sharkey T, Suvachittanont W. Molecular cloning and characterization of two cDNAs encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Hevea brasiliensis . Plant Physiol 2008; 165: 991-1002
  • 34 Chen HL, Lin KW, Gan KH, Wang JP, Won SJ, Lin CN. New diterpenoids and cytotoxic and anti-inflammatory diterpenoids from Amentotaxus formosana . Fitoterapia 2011; 82: 219-224
  • 35 Schardl CL, Byrd AD, Bezion G, Altschuler MA, Hildebrand DF, Hunt AG. Design and construction of a versatile system for the expression of foreign genes in plants. Gene 1987; 61: 1-11
  • 36 Kuzma L, Bruchajer E, Wysolinska H. Methyl jasmonate effect on diterpenoid accumulation in Salvia sclarea hairy root culture in shake flasks and sprinkle bioreactor. Enzyme Microb Technol 2009; 44: 406-410
  • 37 Weigel D, Glazebrook J. Transformation of Agrobacterium using the freeze-thaw method. CSH Protoc 2006; 7: 110-115
  • 38 Murashige T, Skoog F. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 1962; 15: 473-497
  • 39 Doyle JJ, Doyle JL. A rapid total DNA preparation procedure for fresh plant tissue. Focus 1990; 12: 13-15
  • 40 Haas JH, Moore LW, Ream W, Manulis S. Universal PCR primers for detection of phytopathogenic Agrobacterium strains. Appl Environ Microbiol 1995; 61: 2879-2884
  • 41 Bowsher CG, Hucklesby DP, Emes MJ. Nitrite reduction and carbohydrate metabolism in plastids purified from roots of Pisum Sativum L. Planta 1989; 177: 359-366