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Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins

Authors
  • Tzin, Vered1, 2
  • Snyder, John H.1, 3, 4
  • Yang, Dong Sik1, 5
  • Huhman, David V.1
  • Watson, Bonnie S.1
  • Allen, Stacy N.1
  • Tang, Yuhong1
  • Miettinen, Karel6, 7
  • Arendt, Philipp6, 7
  • Pollier, Jacob6, 7
  • Goossens, Alain6, 7
  • Sumner, Lloyd W.1, 8
  • 1 Noble Research Institute, Plant Biology Division, Ardmore, OKLA, 73401, USA , Ardmore (United States)
  • 2 Ben-Gurion University of the Negev, French Associates Institute for Agriculture and Biotechnology of Drylands, Jacob Blaustein Institutes for Desert Research, Sede Boqer Campus, Israel , Sede Boqer Campus (Israel)
  • 3 Cornell University, Department of Plant Biology, Ithaca, NY, 14850, USA , Ithaca (United States)
  • 4 National Institute of Biological Sciences, Beijing, China , Beijing (China)
  • 5 Samsung Advanced Institute of Technology, Biomaterials Laboratory, Material Research Center, Suwon, South Korea , Suwon (South Korea)
  • 6 Ghent University, Department of Plant Biotechnology and Bioinformatics, Ghent, 9052, Belgium , Ghent (Belgium)
  • 7 VIB Center for Plant Systems Biology, Ghent, 9052, Belgium , Ghent (Belgium)
  • 8 University of Missouri, Department of Biochemistry, Interdisciplinary Plant Group, Bond Life Sciences Center, Columbia, MO, 65211, USA , Columbia (United States)
Type
Published Article
Journal
Metabolomics
Publisher
Springer US
Publication Date
May 29, 2019
Volume
15
Issue
6
Identifiers
DOI: 10.1007/s11306-019-1542-1
Source
Springer Nature
Keywords
License
Yellow

Abstract

IntroductionTriterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae.ObjectivesWe characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence.MethodsUHPLC-(-)ESI-QToF-MS was used to profile saponin accumulation across a collection of 106 M. truncatula ecotypes. The profiling results identified numerous ecotypes with high and low saponin accumulation in root and aerial tissues. Four ecotypes with significant differential saponin content in the root and/or aerial tissues were selected, and correlated gene expression profiling was performed.ResultsCorrelation analyses between gene expression and saponin accumulation revealed high correlations between saponin content with gene expression of β-amyrin synthase, MtCYP716A12, and two cytochromes P450 genes, MtCYP72A67 and MtCYP72A68. In vivo and in vitro biochemical assays using yeast microsomes containing MtCYP72A67 revealed hydroxylase activity for carbon 2 of oleanolic acid and hederagenin. This finding was supported by functional characterization of MtCYP72A67 using RNAi-mediated gene silencing in M. truncatula hairy roots, which revealed a significant reduction of 2β-hydroxylated sapogenins. In vivo and in vitro assays with MtCYP72A68 produced in yeast showed multifunctional oxidase activity for carbon 23 of oleanolic acid and hederagenin. These findings were supported by overexpression of MtCYP72A68 in M. truncatula hairy roots, which revealed significant increases of oleanolic acid, 2β-hydroxyoleanolic acid, hederagenin and total saponin levels.ConclusionsThe cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2β-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.

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