Discovery and functional characterization of two diterpene synthases for sclareol biosynthesis in Salvia sclarea(L.) and their relevance for perfume manufacture
Tóm tắt
Sclareol is a diterpene natural product of high value for the fragrance industry. Its labdane carbon skeleton and its two hydroxyl groups also make it a valued starting material for semisynthesis of numerous commercial substances, including production of Ambrox® and related ambergris substitutes used in the formulation of high end perfumes. Most of the commercially-produced sclareol is derived from cultivated clary sage (Salvia sclarea) and extraction of the plant material. In clary sage, sclareol mainly accumulates in essential oil-producing trichomes that densely cover flower calices. Manool also is a minor diterpene of this species and the main diterpene of related Salvia species. Based on previous general knowledge of diterpene biosynthesis in angiosperms, and based on mining of our recently published transcriptome database obtained by deep 454-sequencing of cDNA from clary sage calices, we cloned and functionally characterized two new diterpene synthase (diTPS) enzymes for the complete biosynthesis of sclareol in clary sage. A class II diTPS (SsLPPS) produced labda-13-en-8-ol diphosphate as major product from geranylgeranyl diphosphate (GGPP) with some minor quantities of its non-hydroxylated analogue, (9 S, 10 S)-copalyl diphosphate. A class I diTPS (SsSS) then transformed these intermediates into sclareol and manool, respectively. The production of sclareol was reconstructed in vitro by combining the two recombinant diTPS enzymes with the GGPP starting substrate and in vivo by co-expression of the two proteins in yeast (Saccharomyces cerevisiae). Tobacco-based transient expression assays of green fluorescent protein-fusion constructs revealed that both enzymes possess an N-terminal signal sequence that actively targets SsLPPS and SsSS to the chloroplast, a major site of GGPP and diterpene production in plants. SsLPPS and SsSS are two monofunctional diTPSs which, together, produce the diterpenoid specialized metabolite sclareol in a two-step process. They represent two of the first characterized hydroxylating diTPSs in angiosperms and generate the dihydroxylated labdane sclareol without requirement for additional enzymatic oxidation by activities such as cytochrome P450 monoxygenases. Yeast-based production of sclareol by co-expresssion of SsLPPS and SsSS was efficient enough to warrant the development and use of such technology for the biotechnological production of scareol and other oxygenated diterpenes.
Tài liệu tham khảo
Peters RJ: Two rings in them all: the labdane-related diterpenoids. Nat Prod Rep. 2010, 27: 1521-1530. 10.1039/c0np00019a.
Fleet CM, Sun T: A DELLAcate balance: the role of gibberellin in plant morphogenesis. Curr Opin Plant Biol. 2005, 8: 77-85. 10.1016/j.pbi.2004.11.015.
Yamaguchi S, Sun TP, Kawaide H, Kamiya Y: The GA2 locus of Arabidopsis thaliana encodes ent-kaurene synthase of gibberellin biosynthesis. Plant Physiol. 1998, 116: 1271-1278. 10.1104/pp.116.4.1271.
Bömke C, Tudzynski B: Diversity, regulation, and evolution of the gibberellin biosynthetic pathway in fungi compared to plants and bacteria. Phytochemistry. 2009, 70: 1876-1893. 10.1016/j.phytochem.2009.05.020.
Peters RJ: Uncovering the complex metabolic network underlying diterpenoid phytoalexin biosynthesis in rice and other cereal crop plants. Phytochemistry. 2006, 67: 2307-2317. 10.1016/j.phytochem.2006.08.009.
Keeling CI, Bohlmann J: Genes, enzymes and chemicals of terpenoid diversity in the constitutive and induced defence of conifers against insects and pathogens. New Phytol. 2006, 170: 657-675. 10.1111/j.1469-8137.2006.01716.x.
Keeling CI, Bohlmann J: Diterpene resin acids in conifers. Phytochemistry. 2006, 67: 2415-2423. 10.1016/j.phytochem.2006.08.019.
Gershenzon J, Dudareva N: The function of terpene natural products in the natural world. Nat Chem Biol. 2007, 3: 408-414. 10.1038/nchembio.2007.5.
Bohlmann J, Keeling CI: Terpenoid biomaterials. Plant J. 2008, 54: 656-669. 10.1111/j.1365-313X.2008.03449.x.
Hillwig ML, Mann FM, Peters RJ: Diterpenoid biopolymers: new directions for renewable materials engineering. Biopolymers. 2011, 95: 71-76. 10.1002/bip.21538.
Lawrence BM: Progress in essential oils. Perfumer & Flavorist. 1986, 21: 57-68.
Demetzos C, Stahl B, Anastassaki T, Gazouli M, Tzouvelekis LS, Rallis M: Chemical analysis and antimicrobial activity of the resin Ladano, of its essential oil and of the isolated compounds. Planta Med. 1999, 65: 76-78. 10.1055/s-2006-960444.
Guo Z, Wagner G: Biosynthesis of labdenediol and sclareol in cell-free extracts from trichomes of Nicotiana glutinosa. Planta. 1995, 197: 627-632.
McNeil MJ, Porter RBR, Williams LAD, Rainford L: Chemical composition and antimicrobial activity of the essential oils from Cleome spinosa. Nat Prod Commun. 2010, 5: 1301-1306.
Ulubelen A, Topcu G, Eriş C, Sönmez U, Kartal M, Kurucu S, Bozok-Johansson C: Terpenoids from Salvia sclarea. Phytochemistry. 1994, 36: 971-974. 10.1016/S0031-9422(00)90474-6.
Cutler HG: Plant growth inhibiting properties of diterpenes from tobacco. Plant Cell Physiol. 1977, 18: 711-714.
Bailey JA, Vincent GG, Burden RS: Diterpenes from Nicotiana glutinosa and their Effect on Fungal Growth. Microbiology. 1974, 85: 57-64.
Kennedy BS, Nielsen MT, Severson RF, Sisson VA, Stephenson MK, Jackson DM: Leaf surface chemicals fromNicotiana affecting germination of Peronospora tabacina (adam) sporangia. J Chem Ecol. 1992, 18: 1467-1479. 10.1007/BF00993221.
Chinou I: Labdanes of natural origin-biological activities (1981–2004). Curr Med Chem. 2005, 12: 1295-1317. 10.2174/0929867054020990.
Schmiderer C, Grassi P, Novak J, Weber M, Franz C: Diversity of essential oil glands of clary sage (Salvia sclarea L., Lamiaceae). Plant Biol. 2008, 10: 433-440. 10.1111/j.1438-8677.2008.00053.x.
Barrero AF, Alvarez-Manzaneda EJ, Altarejos J, Salido S, Ramos JM: Synthesis of Ambrox® from (−)-sclareol and (+)-cis-abienol. Tetrahedron. 1993, 49: 10405-10412. 10.1016/S0040-4020(01)80567-6.
Kuźma Ł, Skrzypek Z, Wysokińska H: Diterpenoids and triterpenoids in hairy roots of Salvia sclarea. Plant Cell Tissue Organ Cult. 2005, 84: 100152-100160.
Xu M, Wilderman PR, Morrone D, Xu J, Roy A, Margis-Pinheiro M, Upadhyaya NM, Coates RM, Peters RJ: Functional characterization of the rice kaurene synthase-like gene family. Phytochemistry. 2007, 68: 312-326. 10.1016/j.phytochem.2006.10.016.
Gao W, Hillwig ML, Huang L, Cui G, Wang X, Kong J, Yang B, Peters RJ: A functional genomics approach to tanshinone biosynthesis provides stereochemical insights. Org Lett. 2009, 11: 5170-5173. 10.1021/ol902051v.
Falara V, Pichersky E, Kanellis AK: A copal-8-ol diphosphate synthase from the angiosperm Cistus creticus subsp. creticus is a putative key enzyme for the formation of pharmacologically active, oxygen-containing labdane-type diterpenes. Plant Physiol. 2010, 154: 301-310. 10.1104/pp.110.159566.
Keeling CI, Madilao LL, Zerbe P, Dullat HK, Bohlmann J: The primary diterpene synthase products of Picea abies levopimaradiene/abietadiene synthase (PaLAS) are epimers of a thermally unstable diterpenol. J Biol Chem. 2011, 286: 21145-21153. 10.1074/jbc.M111.245951.
Mafu S, Hillwig ML, Peters RJ: A novel labda-7,13e-dien-15-ol-producing bifunctional diterpene synthase from Selaginella moellendorffii. ChemBioChem. 2011, 12: 1984-1987. 10.1002/cbic.201100336.
Hayashi K-I, Kawaide H, Notomi M, Sakigi Y, Matsuo A, Nozaki H: Identification and functional analysis of bifunctional ent-kaurene synthase from the moss Physcomitrella patens. FEBS Lett. 2006, 580: 6175-6181. 10.1016/j.febslet.2006.10.018.
Ro D-K, Arimura G-I, Lau SYW, Piers E, Bohlmann J: Loblolly pine abietadienol/abietadienal oxidase PtAO (CYP720B1) is a multifunctional, multisubstrate cytochrome P450 monooxygenase. Proc Natl Acad Sci USA. 2005, 102: 8060-8065. 10.1073/pnas.0500825102.
Hamberger B, Ohnishi T, Hamberger B, Séguin A, Bohlmann J: Evolution of diterpene metabolism: Sitka spruce CYP720B4 catalyzes multiple oxidations in resin acid biosynthesis of conifer defense against insects. Plant Physiol. 2011, 157: 1677-1695. 10.1104/pp.111.185843.
Miyazaki S, Katsumata T, Natsume M, Kawaide H: The CYP701B1 of Physcomitrella patens is an ent-kaurene oxidase that resists inhibition by uniconazole-P. FEBS Lett. 2011, 585: 1879-1883. 10.1016/j.febslet.2011.04.057.
Wu Y, Hillwig ML, Wang Q, Peters RJ: Parsing a multifunctional biosynthetic gene cluster from rice: Biochemical characterization of CYP71Z6 & 7. FEBS Lett. 2011, 585: 3446-3451. 10.1016/j.febslet.2011.09.038.
Wang Q, Hillwig ML, Peters RJ: CYP99A3: functional identification of a diterpene oxidase from the momilactone biosynthetic gene cluster in rice. Plant J. 2011, 65: 87-95. 10.1111/j.1365-313X.2010.04408.x.
Morrone D, Chen X, Coates RM, Peters RJ: Characterization of the kaurene oxidase CYP701A3, a multifunctional cytochrome P450 from gibberellin biosynthesis. Biochem J. 2010, 431: 337-344.
Swaminathan S, Morrone D, Wang Q, Fulton DB, Peters RJ: CYP76M7 is an ent-cassadiene C11alpha-hydroxylase defining a second multifunctional diterpenoid biosynthetic gene cluster in rice. Plant Cell. 2009, 21: 3315-3325. 10.1105/tpc.108.063677.
Schalk M: Method for Producing Sclareol. World Intellectual Property organization: Geneva, Switzerland; 2009. Patent No. WO 2009/101126 Al, August 20th, 2009
Kawaide H, Hayashi K, Kawanabe R, Sakigi Y, Matsuo A, Natsume M, Nozaki H: Identification of the single amino acid involved in quenching the ent-kauranyl cation by a water molecule in ent-kaurene synthase of Physcomitrella patens. FEBS J. 2011, 278: 123-133. 10.1111/j.1742-4658.2010.07938.x.
Legrand S, Valot N, Nicolé F, Moja S, Baudino S, Jullien F, Magnard J-L, Caissard J-C, Legendre L: One-step identification of conserved miRNAs, their targets, potential transcription factors and effector genes of complete secondary metabolism pathways after 454 pyrosequencing of calyx cDNAs from the Labiate Salvia sclarea L. Gene. 2010, 450: 55-62. 10.1016/j.gene.2009.10.004.
Chen F, Tholl D, Bohlmann J, Pichersky E: The family of terpene synthases in plants: a mid-size family of genes for specialized metabolism that is highly diversified throughout the kingdom. Plant J. 2011, 66: 212-229. 10.1111/j.1365-313X.2011.04520.x.
Bohlmann J, Meyer-Gauen G, Croteau R: Plant terpenoid synthases: molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA. 1998, 95: 4126-4133. 10.1073/pnas.95.8.4126.
Köksal M, Hu H, Coates RM, Peters RJ, Christianson DW: Structure and mechanism of the diterpene cyclase ent-copalyl diphosphate synthase. Nat Chem Biol. 2011, 7: 431-433. 10.1038/nchembio.578.
Zhou K, Gao Y, Hoy JA, Mann FM, Honzatko RB, Peters RJ: Insights into diterpene cyclization from the structure of the bifunctional abietadiene synthase from Abies grandis. J Biol Chem. 2012, 287: 6840-6850. 10.1074/jbc.M111.337592.
Hillwig ML, Xu M, Toyomasu T, Tiernan MS, Wei G, Cui G, Huang L, Peters RJ: Domain loss has independently occurred multiple times in plant terpene synthase evolution. Plant J. 2011, 68: 1051-1060. 10.1111/j.1365-313X.2011.04756.x.
Cao R, Zhang Y, Mann FM, Huang C, Mukkamala D, Hudock MP, Mead ME, Prisic S, Wang K, Lin F-Y, Chang T-K, Peters RJ, Oldfield E: Diterpene cyclases and the nature of the isoprene fold. Proteins. 2010, 78: 2417-2432. 10.1002/prot.22751.
Harris LJ, Saparno A, Johnston A, Prisic S, Xu M, Allard S, Kathiresan A, Ouellet T, Peters RJ: The maize An2 gene is induced by Fusarium attack and encodes an ent-copalyl diphosphate synthase. Plant Mol Biol. 2005, 59: 881-894. 10.1007/s11103-005-1674-8.
Salimpour F: Chemotaxonomy of six Salvia species using essential oil composition markers. J Med Plants Res. 2011, 5: 1795-1805.
Couladis M, Tzakou O, Stojanovic D, Mimica-Dukic N, Jancic R: The essential oil composition of Salvia argentea L. Flavour Frag J. 2001, 16: 227-229. 10.1002/ffj.989.
Zhou YJ, Gao W, Rong Q, Jin G, Chu H, Liu W, Yang W, Zhu Z, Li G, Zhu G, Huang L, Zhao ZK: Modular Pathway Engineering of Diterpenoid Synthases and the Mevalonic Acid Pathway for Miltiradiene Production. J Am Chem Soc. 2012, 134: 3234-3241.
Zhou K, Peters RJ: Investigating the conservation pattern of a putative second terpene synthase divalent metal binding motif in plants. Phytochemistry. 2009, 70: 366-369. 10.1016/j.phytochem.2008.12.022.
Christianson DW: Structural biology and chemistry of the terpenoid cyclases. Chem Rev. 2006, 106: 3412-3442. 10.1021/cr050286w.
Ajikumar PK, Xiao W-H, Tyo KEJ, Wang Y, Simeon F, Leonard E, Mucha O, Phon TH, Pfeifer B, Stephanopoulos G: Isoprenoid pathway optimization for taxol precursor overproduction in Escherichia coli. Science. 2010, 330: 70-74. 10.1126/science.1191652.
Westfall PJ, Pitera DJ, Lenihan JR, Eng D, Woolard FX, Regentin R, Horning T, Tsuruta H, Melis DJ, Owens A, Fickes S, Diola D, Benjamin KR, Keasling JD, Leavell MD, McPHee DJ, Renninger NS, Newman JD, Paddon CJ: Production of amorphadiene in yeast, and its conversion to dihydroartemisinic acid, precursor to the antimalarial agent artemisinin. Proc Nat Am Soc. 2011, 109: E111-E118.
Guindon S, Dufayard J-F, Lefort V, Anisimova M, Hordijk W, Gascuel O: New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010, 59: 307-321. 10.1093/sysbio/syq010.
Page RD: TreeView: an application to display phylogenetic trees on personal computers. Comput Appl Biosci. 1996, 12: 357-358.
Miroux B, Walker JE: Over-production of proteins in Escherichia coli: mutant hosts that allow synthesis of some membrane proteins and globular proteins at high levels. J Mol Biol. 1996, 260: 289-298. 10.1006/jmbi.1996.0399.
Zerbe P, Chiang A, Bohlmann J: Mutational analysis of white spruce (Picea glauca) ent-kaurene synthase (PgKS) reveals common and distinct mechanisms of conifer diterpene synthases of general and specialized metabolism. Phytochemistry. 2012, 74: 30-39.
Keeling CI, Weisshaar S, Lin RPC, Bohlmann J: Functional plasticity of paralogous diterpene synthases involved in conifer defense. Proc Natl Acad Sci USA. 2008, 105: 1085-1090. 10.1073/pnas.0709466105.
Ro D-K, Ehlting J, Douglas CJ: Cloning, functional expression, and subcellular localization of multiple NADPH-cytochrome P450 reductases from hybrid poplar. Plant Physiol. 2002, 130: 1837-1851. 10.1104/pp.008011.
Xu R, Li QQ: Protocol: Streamline cloning of genes into binary vectors in Agrobacterium via the Gateway(R) TOPO vector system. Plant Methods. 2008, 4: 4-10.1186/1746-4811-4-4.
Bassard J-E, Mutterer J, Duval F, Werck-Reichhart D: A novel method for monitoring the localization of cytochromes P450 and other endoplasmic reticulum membrane associated proteins: a tool for investigating the formation of metabolons. FEBS J. 2012, 279: 1576-1583. 10.1111/j.1742-4658.2011.08312.x.
Voinnet O, Rivas S, Mestre P, Baulcombe D: An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 2003, 33: 949-956. 10.1046/j.1365-313X.2003.01676.x.
Abramoff MD: Image Processing with ImageJ. Biophoton Int. 2004, 11: 36-42.