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

(2019) METABOLOMICS. 15(6).
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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 -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 2-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, 2-hydroxyoleanolic acid, hederagenin and total saponin levels. Conclusions: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.
Keywords
Saponin, Sapogenin, Cytochrome P450, CYP72A67, CYP72A68, Medicago truncatula, Integrated metabolomics, TRITERPENE SAPONIN BIOSYNTHESIS, MINIMUM REPORTING STANDARDS, GENE-EXPRESSION ATLAS, BETA-AMYRIN, COMBINATORIAL BIOSYNTHESIS, NATURAL-PRODUCTS, PROANTHOCYANIDIN BIOSYNTHESIS, BIOLOGICAL-ACTIVITIES, YEAST EXPRESSION, ALFALFA SAPONINS

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Citation

Please use this url to cite or link to this publication:

MLA
Tzin, Vered et al. “Integrated Metabolomics Identifies CYP72A67 and CYP72A68 Oxidases in the Biosynthesis of Medicago Truncatula Oleanate Sapogenins.” METABOLOMICS 15.6 (2019): n. pag. Print.
APA
Tzin, V., Snyder, J. H., Yang, D. S., Huhman, D. V., Watson, B. S., Allen, S. N., Tang, Y., et al. (2019). Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins. METABOLOMICS, 15(6).
Chicago author-date
Tzin, Vered, John H Snyder, Dong Sik Yang, David V Huhman, Bonnie S Watson, Stacy N Allen, Yuhong Tang, et al. 2019. “Integrated Metabolomics Identifies CYP72A67 and CYP72A68 Oxidases in the Biosynthesis of Medicago Truncatula Oleanate Sapogenins.” Metabolomics 15 (6).
Chicago author-date (all authors)
Tzin, Vered, 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, and Lloyd W Sumner. 2019. “Integrated Metabolomics Identifies CYP72A67 and CYP72A68 Oxidases in the Biosynthesis of Medicago Truncatula Oleanate Sapogenins.” Metabolomics 15 (6).
Vancouver
1.
Tzin V, Snyder JH, Yang DS, Huhman DV, Watson BS, Allen SN, et al. Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins. METABOLOMICS. 2019;15(6).
IEEE
[1]
V. Tzin et al., “Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins,” METABOLOMICS, vol. 15, no. 6, 2019.
@article{8617951,
  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 -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 2-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, 2-hydroxyoleanolic acid, hederagenin and total saponin levels.
Conclusions: The cumulative data support that MtCYP72A68 is a multisubstrate, multifunctional oxidase and MtCYP72A67 is a 2-hydroxylase, both of which function during the early steps of triterpene-oleanate sapogenin biosynthesis.},
  articleno    = {85},
  author       = {Tzin, Vered and Snyder, John H and Yang, Dong Sik and Huhman, David V and Watson, Bonnie S and Allen, Stacy N and Tang, Yuhong and Miettinen, Karel and Arendt, Philipp and Pollier, Jacob and Goossens, Alain and Sumner, Lloyd W},
  issn         = {1573-3882},
  journal      = {METABOLOMICS},
  keywords     = {Saponin,Sapogenin,Cytochrome P450,CYP72A67,CYP72A68,Medicago truncatula,Integrated metabolomics,TRITERPENE SAPONIN BIOSYNTHESIS,MINIMUM REPORTING STANDARDS,GENE-EXPRESSION ATLAS,BETA-AMYRIN,COMBINATORIAL BIOSYNTHESIS,NATURAL-PRODUCTS,PROANTHOCYANIDIN BIOSYNTHESIS,BIOLOGICAL-ACTIVITIES,YEAST EXPRESSION,ALFALFA SAPONINS},
  language     = {eng},
  number       = {6},
  pages        = {20},
  title        = {Integrated metabolomics identifies CYP72A67 and CYP72A68 oxidases in the biosynthesis of Medicago truncatula oleanate sapogenins},
  url          = {http://dx.doi.org/10.1007/s11306-019-1542-1},
  volume       = {15},
  year         = {2019},
}

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