Overexpression and RNAi-mediated Knockdown of Two 3β-hydroxy-Δ5-steroid dehydrogenase Genes in Digitalis lanata Shoot Cultures Reveal Their Role in Cardenolide Biosynthesis^[*]

Abstract

3β-hydroxy-Δ5-steroid dehydrogenases (3βHSDs) are supposed to be involved in 5β-cardenolide biosynthesis. Here, a novel 3βHSD (Dl3βHSD2) was isolated from Digitalis lanata shoot cultures and expressed in E. coli. Recombinant Dl3βHSD1 and Dl3βHSD2 shared 70% amino acid identity, reduced various 3-oxopregnanes and oxidised 3-hydroxypregnanes, but only rDl3βHSD2 converted small ketones and secondary alcohols efficiently. To explain these differences in substrate specificity, we established homology models using borneol dehydrogenase of Salvia rosmarinus (6zyz) as the template. Hydrophobicity and amino acid residues in the binding pocket may explain the difference in enzyme activities and substrate preferences. Compared to Dl3βHSD1, Dl3βHSD2 is weakly expressed in D. lanata shoots. High constitutive expression of Dl3βHSDs was realised by Agrobacterium-mediated transfer of Dl3βHSD genes fused to the CaMV-35S promotor into the genome of D. lanata wild type shoot cultures. Transformed shoots (35S:Dl3βHSD1 and 35S:Dl3βHSD2) accumulated less cardenolides than controls. The levels of reduced glutathione (GSH), which is known to inhibit cardenolide formation, were higher in the 35S:Dl3βHSD1 lines than in the controls. In the 35S:Dl3βHSD1 lines cardenolide levels were restored after adding of the substrate pregnane-3,20-dione in combination with buthionine-sulfoximine (BSO), an inhibitor of GSH formation. RNAi-mediated knockdown of the Dl3βHSD1 yielded several shoot culture lines with strongly reduced cardenolide levels. In these lines, cardenolide biosynthesis was fully restored after addition of the downstream precursor pregnan-3β-ol-20-one, whereas upstream precursors such as progesterone had no effect, indicating that no shunt pathway could overcome the Dl3βHSD1 knockdown. These results can be taken as the first direct proof that Dl3βHSD1 is indeed involved in 5β-cardenolide biosynthesis.

Key words

Digitalis lanata - Plantaginaceae - cardenolide biosynthesis - 3β-hydroxy-Δ5-steroid dehydrogenase - RNAi-mediated gene knockdown

^* Dedicated to Prof. Gerhard Franz, University of Regensburg, Germany, on the occasion of his 85th birthday.

Publication History

Received: 11 November 2022

Accepted after revision: 31 March 2023

Article published online:
15 May 2023

• References

• 1 Brown L, Lorenz B, Erdmann E. Reduced positive inotropic effects in diseased human ventricular myocardium. Cardiovasc Res 1986; 20: 516-520 DOI: 10.1093/cvr/20.7.516.
• 2 Schneider NFZ, Cerella C, Simões CMO, Diederich M. Anticancer and immunogenic properties of cardiac glycosides. Molecules 2017; 22: 1932 DOI: 10.3390/molecules22111932.
• 3 Kreis W. The Foxgloves (Digitalis) Revisited. Planta Med 2017; 83: 962-976 DOI: 10.1055/s-0043-111240.
• 4 Rieck C, Geiger D, Munkert J, Messerschmidt K, Petersen J, Strasser J, Meitinger N, Kreis W. Biosynthetic approach to combine the first steps of cardenolide formation in Saccharomyces cerevisiae . Microbiologyopen 2019; 8: e925 DOI: 10.1002/mbo3.925.
• 5 Gärtner DE, Keilholz W, Seitz HU. Purification, characterization and partial peptide microsequencing of progesterone 5 beta-reductase from shoot cultures of Digitalis purpurea . Eur J Biochem 1994; 225: 1125-1132 DOI: 10.1111/j.1432-1033.1994.1125b.x.
• 6 Klein J, Horn E, Ernst M, Leykauf T, Leupold T, Dorfner M, Wolf L, Ignatova A, Kreis W, Munkert J. RNAi-mediated gene knockdown of progesterone 5β-reductases in Digitalis lanata reduces 5β-cardenolide content. Plant Cell Rep 2021; 40: 1631-1646 DOI: 10.1007/s00299-021-02707-3.
• 7 Seidel S, Kreis W, Reinhard E. Δ5-3β-hydroxysteroid dehydrogenase/Δ5-Δ4-ketosteroid isomerase (3β-HSD), a possible enzyme of cardiac glycoside biosynthesis, in cell cultures and plants of Digitalis lanata EHRH. Plant Cell Rep 1990; 8: 621-624 DOI: 10.1007/BF00270068.
• 8 Finsterbusch A, Lindemann P, Grimm R, Eckerskorn C, Luckner M. Delta(5)-3beta-hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism?. Planta 1999; 209: 478-486 DOI: 10.1007/s004250050751.
• 9 Persson B, Kallberg Y, Bray JE, Bruford E, Dellaporta SL, Favia AD, Duarte RG, Jörnvall H, Kavanagh KL, Kedishvili N, Kisiela M, Maser E, Mindnich R, Orchard S, Penning TM, Thornton JM, Adamski J, Oppermann U. The SDR (short-chain dehydrogenase/reductase and related enzymes) nomenclature initiative. Chem Biol Interact 2009; 178: 94-98 DOI: 10.1016/j.cbi.2008.10.040.
• 10 Kallberg Y, Oppermann U, Jörnvall H, Persson B. Short-chain dehydrogenases/reductases (SDRs). Eur J Biochem 2002; 269: 4409-4417 DOI: 10.1046/j.1432-1033.2002.03130.x.
• 11 Ernst M, de Padua RM, Herl V, Müller-Uri F, Kreis W. Expression of 3beta-HSD and P5betaR, genes respectively coding for Delta5-3beta-hydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of Digitalis lanata EHRH. Planta Med 2010; 76: 923-927 DOI: 10.1055/s-0030-1250007.
• 12 Munkert J, Ernst M, Müller-Uri F, Kreis W. Identification and stress-induced expression of three 3β-hydroxysteroid dehydrogenases from Erysimum crepidifolium Rchb. and their putative role in cardenolide biosynthesis. Phytochemistry 2014; 100: 26-33 DOI: 10.1016/j.phytochem.2014.01.006.
• 13 Herl V, Fischer G, Reva VA, Stiebritz M, Muller YA, Müller-Uri F, Kreis W. The VEP1 gene (At4g24220) encodes a short-chain dehydrogenase/reductase with 3-oxo-Δ4, 5-steroid 5β-reductase activity in Arabidopsis thaliana L. Biochimie 2009; 91: 517-525 DOI: 10.1016/j.biochi.2008.12.005.
• 14 Klein J, Ernst M, Christmann A, Tropper M, Leykauf T, Kreis W, Munkert J. Knockout of Arabidopsis thaliana VEP1, encoding a PRISE (progesterone 5β-reductase/iridoid synthase-like enzyme), leads to metabolic changes in response to exogenous Methyl Vinyl Ketone (MVK). Metabolites 2021; 12: 11 DOI: 10.3390/metabo12010011.
• 15 Nguyen TD, OʼConnor SE. The progesterone 5β-reductase/iridoid synthase family: A catalytic reservoir for specialized metabolism across land plants. ACS Chem Biol 2020; 15: 1780-1787 DOI: 10.1021/acschembio.0c00220.
• 16 Munkert J, Costa C, Budeanu O, Petersen J, Bertolucci S, Fischer G, Müller-Uri F, Kreis W. Progesterone 5β-reductase genes of the Brassicaceae family as function-associated molecular markers. Plant Biol (Stuttg) 2015; 17: 1113-1122 DOI: 10.1111/plb.12361.
• 17 Rahier A, Darnet S, Bouvier F, Camara B, Bard M. Molecular and enzymatic characterizations of novel bifunctional 3beta-hydroxysteroid dehydrogenases/C-4 decarboxylases from Arabidopsis thaliana . J Biol Chem 2006; 281: 27264-27277 DOI: 10.1074/jbc.M604431200.
• 18 Lee HJ, Nakayasu M, Akiyama R, Kobayashi M, Miyachi H, Sugimoto Y, Umemoto N, Saito K, Muranaka T, Mizutani M. Identification of a 3β-hydroxysteroid dehydrogenase/3-ketosteroid reductase involved in α-tomatine biosynthesis in tomato. Plant Cell Physiol 2019; 60: 1304-1315 DOI: 10.1093/pcp/pcz049.
• 19 Meitinger N, Munkert J, Maia de Pádua R, de Souza Filho JD, Maid H, Bauer W, Braga FC, Kreis W. The catalytic mechanism of the 3-ketosteroid isomerase of Digitalis lanata involves an intramolecular proton transfer and the activity is not associated with the 3β-hydroxysteroid dehydrogenase activity. Tetrahedron Lett 2016; 57: 1567-1571 DOI: 10.1016/j.tetlet.2016.02.099.
• 20 Herl V, Frankenstein J, Meitinger N, Müller-Uri F, Kreis W. Delta 5-3beta-hydroxysteroid dehydrogenase (3 beta HSD) from Digitalis lanata. Heterologous expression and characterisation of the recombinant enzyme. Planta Med 2007; 73: 704-710 DOI: 10.1055/s-2007-981537.
• 21 Tropper M, Höhn S, Wolf LS, Fritsch J, Kastner-Detter N, Rieck C, Munkert J, Meitinger N, Lanig H, Kreis W. 21-Hydroxypregnane 21-O-malonylation, a crucial step in cardenolide biosynthesis, can be achieved by substrate-promiscuous BAHD-type phenolic glucoside malonyltransferases from Arabidopsis thaliana and homolog proteins from Digitalis lanata . Phytochemistry 2021; 187: 112710 DOI: 10.1016/j.phytochem.2021.112710.
• 22 Sellés Vidal L, Kelly CL, Mordaka PM, Heap JT. Review of NAD(P)H-dependent oxidoreductases: Properties, engineering and application. Biochim Biophys Acta Proteins Proteom 2018; 1866: 327-347 DOI: 10.1016/j.bbapap.2017.11.005.
• 23 Waterhouse A, Bertoni M, Bienert S, Studer G, Tauriello G, Gumienny R, Heer FT, de Beer TAP, Rempfer C, Bordoli L, Lepore R, Schwede T. SWISS-MODEL: Homology modelling of protein structures and complexes. Nucleic Acids Res 2018; 46: W296-W303 DOI: 10.1093/nar/gky427.
• 24 Chánique AM, Dimos N, Drienovská I, Calderini E, Pantín MP, Helmer CPO, Hofer M, Sieber V, Parra LP, Loll B, Kourist R. A structural view on the stereospecificity of plant borneol-type dehydrogenases. ChemCatChem 2021; 13: 2262-2277 DOI: 10.1002/cctc.202100110.
• 25 Trott O, Olson AJ. AutoDock Vina: Improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J Comput Chem 2010; 31: 455-461 DOI: 10.1002/jcc.21334.
• 26 Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A. Protein Identification and Analysis Tools on the ExPASy Server. In: Walker JM. ed. The Proteomics Protocols Handbook. Totowa, NJ: Humana Press; 2005: 571-607
• 27 Kyte J, Doolittle RF. A simple method for displaying the hydropathic character of a protein. J Mol Biol 1982; 157: 105-132 DOI: 10.1016/0022-2836(82)90515-0.
• 28 Kairuz E, Pérez-Alonso N, Capote-Pérez A, Pérez-Pérez A, Espinosa-Antón AA, Angenon G, Jiménez E, Chong-Pérez B. Enhancement of cardenolide production in transgenic Digitalis purpurea L. by expressing a progesterone-5β-reductase from Arabidopsis thaliana L. Ind Crops Prod 2020; 146: 112166 DOI: 10.1016/j.indcrop.2020.112166.
• 29 de Beuckeleer M, Lemmers M, de Vos G, Willmitzer L, van Montagu M, Schell J. Further insight on the transferred-DNA of octopine crown gall. Mol Gen Genet 1981; 183: 283-288 DOI: 10.1007/BF00270630.
• 30 Eisenbeiß M, Kreis W, Reinhard E. Cardenolide biosynthesis in light- and dark-grown Digitalis lanata shoot cultures. Plant Physiology and Biochemistry 1999; 37: 13-23 DOI: 10.1016/S0981-9428(99)80062-X.
• 31 Mirzaei M, Züst T, Younkin GC, Hastings AP, Alani ML, Agrawal AA, Jander G. Less is more: A mutation in the chemical defense pathway of Erysimum cheiranthoides (Brassicaceae) reduces total cardenolide abundance but increases resistance to insect herbivores. J Chem Ecol 2020; 46: 1131-1143 DOI: 10.1007/s10886-020-01225-y.
• 32 Kreis W, Munkert J. Exploiting enzyme promiscuity to shape plant specialized metabolism. J Exp Bot 2019; 70: 1435-1445 DOI: 10.1093/jxb/erz025.
• 33 Anterola AM, Lewis NG. Trends in lignin modification: A comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry 2002; 61: 221-294 DOI: 10.1016/S0031-9422(02)00211-X.
• 34 Verpoorte R, Contin A, Memelink J. Biotechnology for the production of plant secondary metabolites. Phytochemistry Reviews 2002; 1: 13-25 DOI: 10.1023/A:1015871916833.
• 35 Mahmoud SS, Williams M, Croteau R. Cosuppression of limonene-3-hydroxylase in peppermint promotes accumulation of limonene in the essential oil. Phytochemistry 2004; 65: 547-554 DOI: 10.1016/j.phytochem.2004.01.005.
• 36 OʼConnor SE. Engineering of Secondary Metabolism. Annu Rev Genet 2015; 49: 71-94 DOI: 10.1146/annurev-genet-120213-092053.
• 37 Zhao Q, Dixon RA. Transcriptional networks for lignin biosynthesis: More complex than we thought?. Trends Plant Sci 2011; 16: 227-233 DOI: 10.1016/j.tplants.2010.12.005.
• 38 Bruegmann T, Wetzel H, Hettrich K, Smeds A, Willför S, Kersten B, Fladung M. Knockdown of PCBER1, a gene of neolignan biosynthesis, resulted in increased poplar growth. Planta 2019; 249: 515-525 DOI: 10.1007/s00425-018-3021-8.
• 39 Goklany S, Loring RH, Glick J, Lee-Parsons CWT. Assessing the limitations to terpenoid indole alkaloid biosynthesis in Catharanthus roseus hairy root cultures through gene expression profiling and precursor feeding. Biotechnol Prog 2009; 25: 1289-1296 DOI: 10.1002/btpr.204.
• 40 Berglund T, Ohlsson AB. The glutathione biosynthesis inhibitor Buthionine-Sulfoximine (BSO) induces cardenolide accumulation in Digitalis lanata tissue culture. J Plant Physiol 1993; 142: 248-250 DOI: 10.1016/S0176-1617(11)80973-9.
• 41 Li N, Zeng W, Xu S, Zhou J. Toward fine-tuned metabolic networks in industrial microorganisms. Synth Syst Biotechnol 2020; 5: 81-91 DOI: 10.1016/j.synbio.2020.05.002.
• 42 Wagner GJ, Kroumova AB. The Use of RNAi to Elucidate and Manipulate Secondary Metabolite Synthesis in Plants. In: Ying SY. ed. Current Perspectives in microRNAs (miRNA). Dordrecht: Springer Netherlands; 2008: 431-459
• 43 Munkert J, Pollier J, Miettinen K, van Moerkercke A, Payne R, Müller-Uri F, Burlat V, OʼConnor SE, Memelink J, Kreis W, Goossens A. Iridoid synthase activity is common among the plant progesterone 5β-reductase family. Mol Plant 2015; 8: 136-152 DOI: 10.1016/j.molp.2014.11.005.
• 44 Taliansky M, Samarskaya V, Zavriev SK, Fesenko I, Kalinina NO, Love AJ. RNA-based technologies for engineering plant virus resistance. Plants (Basel) 2021; 10: 82 DOI: 10.3390/plants10010082.
• 45 Fujii N, Inui T, Iwasa K, Morishige T, Sato F. Knockdown of berberine bridge enzyme by RNAi accumulates (S)-reticuline and activates a silent pathway in cultured California poppy cells. Transgenic Res 2007; 16: 363-375 DOI: 10.1007/s11248-006-9040-4.
• 46 Haussmann W, Kreis W, Stuhlemmer U, Reinhard E. Effects of various pregnanes and two 23-nor-5-cholenic acids on cardenolide accumulation in cell and organ cultures of Digitalis lanata . Planta Med 1997; 63: 446-453 DOI: 10.1055/s-2006-957731.
• 47 Paper DH, Franz G. Biotransformation of 5 βH-pregnan-3βol-20-one and cardenolides in cell suspension cultures of Nerium oleander L. Plant Cell Rep 1990; 8: 651-655 DOI: 10.1007/BF00269985.
• 48 Berman H, Henrick K, Nakamura H. Announcing the worldwide Protein Data Bank. Nat Struct Biol 2003; 10: 980 DOI: 10.1038/nsb1203-980.
• 49 Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE. UCSF Chimera–a visualization system for exploratory research and analysis. J Comput Chem 2004; 25: 1605-1612 DOI: 10.1002/jcc.20084.
• 50 Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 2008; 3: 1101-1108 DOI: 10.1038/nprot.2008.73.
• 51 Crooks GE, Hon G, Chandonia JM, Brenner SE. WebLogo: A sequence logo generator. Figure 1. Genome Res 2004; 14: 1188-1190 DOI: 10.1101/gr.849004.