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

Vered Tzin; John H. Snyder; Dong Sik Yang; David V. Huhman; Bonnie S. Watson; Stacy N. Allen; Yuhong Tang; Karel Miettinen; Philipp Arendt; Jacob Pollier; Alain Goossens; Lloyd W. Sumner

doi:10.1007/s11306-019-1542-1

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

Vered Tzin, John H. Snyder, Dong Sik Yang, David V. Huhman, Bonnie S. Watson, Stacy N. Allen, Yuhong Tang, Karel Miettinen, Philipp Arendt, Jacob Pollier, Alain Goossens, Lloyd W. Sumner

The French Associates Institute for Agriculture and Biotechnology of Drylands

Research output: Contribution to journal › Article › peer-review

27 Scopus citations

Abstract

Introduction: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. Objectives: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. Methods: UHPLC-(-)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. Results: Correlation 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. Conclusions: The 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.

Original language	English
Article number	85
Journal	Metabolomics
Volume	15
Issue number	6
DOIs	https://doi.org/10.1007/s11306-019-1542-1
State	Published - 1 Jun 2019

Keywords

CYP72A67
CYP72A68
Cytochrome P450
Integrated metabolomics
Medicago truncatula
Sapogenin
Saponin

ASJC Scopus subject areas

Endocrinology, Diabetes and Metabolism
Biochemistry
Clinical Biochemistry

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10.1007/s11306-019-1542-1

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Tzin, V., Snyder, J. H., Yang, D. S., Huhman, D. V., Watson, B. S., Allen, S. N., Tang, Y., Miettinen, K., Arendt, P., Pollier, J., Goossens, A., & Sumner, L. W. (2019). Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins. Metabolomics, 15(6), Article 85. https://doi.org/10.1007/s11306-019-1542-1

@article{ea1612c068ed4ab4918833ac0f89559d,

title = "Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins",

abstract = "Introduction: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. Objectives: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. Methods: UHPLC-(-)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. Results: Correlation 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. Conclusions: The 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.",

keywords = "CYP72A67, CYP72A68, Cytochrome P450, Integrated metabolomics, Medicago truncatula, Sapogenin, Saponin",

author = "Vered Tzin and Snyder, {John H.} and Yang, {Dong Sik} and Huhman, {David V.} and Watson, {Bonnie S.} and Allen, {Stacy N.} and Yuhong Tang and Karel Miettinen and Philipp Arendt and Jacob Pollier and Alain Goossens and Sumner, {Lloyd W.}",

note = "Funding Information: Funding The Sumner lab has been graciously supported by several entities over the years for the development of natural products profiling and plant metabolomics. This project was financially supported by the Oklahoma Center for the Advancement of Science and Technology (OCAST Award#PSB10-027), the National Science Foundation Molecular and Cellular Biosciences Award#1024976, The Samuel Roberts Noble Foundation, Oklahoma EPSCoR Research Experience for Undergraduates subaward #EPSCoR-2011-15, the VIB International PhD Fellowship Program (predoctoral fellowship to P.A.), the Research Foundation Flanders (postdoctoral fellowship to J.P.), and the European Union Seventh Framework Program FP7/2007–2013 under grant agreement number 613692–TriForC. Funding Information: We thank Dr. Jean-Marie Prosperi and the INRA (http://institut.inra.fr/en) for providing the M. truncatula germplasm collection. We also thank Joseph Chappell for providing the WAT11 yeast strain. We thank Dr. Qiao Zhao for constructive comments on this manuscript. Publisher Copyright: {\textcopyright} 2019, Springer Science+Business Media, LLC, part of Springer Nature.",

year = "2019",

month = jun,

day = "1",

doi = "10.1007/s11306-019-1542-1",

language = "English",

volume = "15",

journal = "Metabolomics",

issn = "1573-3882",

publisher = "Springer New York",

number = "6",

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TY - JOUR

T1 - Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins

AU - Tzin, Vered

AU - Snyder, John H.

AU - Yang, Dong Sik

AU - Huhman, David V.

AU - Watson, Bonnie S.

AU - Allen, Stacy N.

AU - Tang, Yuhong

AU - Miettinen, Karel

AU - Arendt, Philipp

AU - Pollier, Jacob

AU - Goossens, Alain

AU - Sumner, Lloyd W.

N1 - Funding Information: Funding The Sumner lab has been graciously supported by several entities over the years for the development of natural products profiling and plant metabolomics. This project was financially supported by the Oklahoma Center for the Advancement of Science and Technology (OCAST Award#PSB10-027), the National Science Foundation Molecular and Cellular Biosciences Award#1024976, The Samuel Roberts Noble Foundation, Oklahoma EPSCoR Research Experience for Undergraduates subaward #EPSCoR-2011-15, the VIB International PhD Fellowship Program (predoctoral fellowship to P.A.), the Research Foundation Flanders (postdoctoral fellowship to J.P.), and the European Union Seventh Framework Program FP7/2007–2013 under grant agreement number 613692–TriForC. Funding Information: We thank Dr. Jean-Marie Prosperi and the INRA (http://institut.inra.fr/en) for providing the M. truncatula germplasm collection. We also thank Joseph Chappell for providing the WAT11 yeast strain. We thank Dr. Qiao Zhao for constructive comments on this manuscript. Publisher Copyright: © 2019, Springer Science+Business Media, LLC, part of Springer Nature.

PY - 2019/6/1

Y1 - 2019/6/1

N2 - Introduction: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. Objectives: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. Methods: UHPLC-(-)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. Results: Correlation 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. Conclusions: The 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.

AB - Introduction: Triterpene saponins are important bioactive plant natural products found in many plant families including the Leguminosae. Objectives: We characterize two Medicago truncatula cytochrome P450 enzymes, MtCYP72A67 and MtCYP72A68, involved in saponin biosynthesis including both in vitro and in planta evidence. Methods: UHPLC-(-)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. Results: Correlation 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. Conclusions: The 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.

KW - CYP72A67

KW - CYP72A68

KW - Cytochrome P450

KW - Integrated metabolomics

KW - Medicago truncatula

KW - Sapogenin

KW - Saponin

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AN - SCOPUS:85066500300

SN - 1573-3882

VL - 15

JO - Metabolomics

JF - Metabolomics

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ER -

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

Abstract

Keywords

ASJC Scopus subject areas

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