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Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

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Abstract
The saikosaponins comprise oleanane-and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16 alpha hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated beta-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.
Keywords
PANAX-GINSENG, TRITERPENE CYCLIZATION, GINSENOSIDE BIOSYNTHESIS, BETA-AMYRIN SYNTHASE, triterpenoid, metabolic engineering, amyrin, cyclodextrin, MEDICAGO-TRUNCATULA, CYTOCHROME-P450, CYCLODEXTRINS, GLYCYRRHIZIN, LICORICE, PLANTS

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Citation

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MLA
Moses, Tessa, Jacob Pollier, Lorena Almagro, et al. “Combinatorial Biosynthesis of Sapogenins and Saponins in Saccharomyces Cerevisiae Using a C-16α Hydroxylase from Bupleurum Falcatum.” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 111.4 (2014): 1634–1639. Print.
APA
Moses, T., Pollier, J., Almagro, L., Buyst, D., Van Montagu, M., Pedreño, M. A., Martins, J., et al. (2014). Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 111(4), 1634–1639.
Chicago author-date
Moses, Tessa, Jacob Pollier, Lorena Almagro, Dieter Buyst, Marc Van Montagu, María A Pedreño, José Martins, Johan M Thevelein, and Alain Goossens. 2014. “Combinatorial Biosynthesis of Sapogenins and Saponins in Saccharomyces Cerevisiae Using a C-16α Hydroxylase from Bupleurum Falcatum.” Proceedings of the National Academy of Sciences of the United States of America 111 (4): 1634–1639.
Chicago author-date (all authors)
Moses, Tessa, Jacob Pollier, Lorena Almagro, Dieter Buyst, Marc Van Montagu, María A Pedreño, José Martins, Johan M Thevelein, and Alain Goossens. 2014. “Combinatorial Biosynthesis of Sapogenins and Saponins in Saccharomyces Cerevisiae Using a C-16α Hydroxylase from Bupleurum Falcatum.” Proceedings of the National Academy of Sciences of the United States of America 111 (4): 1634–1639.
Vancouver
1.
Moses T, Pollier J, Almagro L, Buyst D, Van Montagu M, Pedreño MA, et al. Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum. PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 2014;111(4):1634–9.
IEEE
[1]
T. Moses et al., “Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum,” PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, vol. 111, no. 4, pp. 1634–1639, 2014.
@article{4349893,
  abstract     = {The saikosaponins comprise oleanane-and ursane-type triterpene saponins that are abundantly present in the roots of the genus Bupleurum widely used in Asian traditional medicine. Here we identified a gene, designated CYP716Y1, encoding a cytochrome P450 monooxygenase from Bupleurum falcatum that catalyzes the C-16 alpha hydroxylation of oleanane- and ursane-type triterpenes. Exploiting this hitherto unavailable enzymatic activity, we launched a combinatorial synthetic biology program in which we combined CYP716Y1 with oxidosqualene cyclase, P450, and glycosyltransferase genes available from other plant species and reconstituted the synthesis of monoglycosylated saponins in yeast. Additionally, we established a culturing strategy in which applying methylated beta-cyclodextrin to the culture medium allows the sequestration of heterologous nonvolatile hydrophobic terpenes, such as triterpene sapogenins, from engineered yeast cells into the growth medium, thereby greatly enhancing productivity. Together, our findings provide a sound base for the development of a synthetic biology platform for the production of bioactive triterpene sapo(ge)nins.},
  author       = {Moses, Tessa and Pollier, Jacob and Almagro, Lorena and Buyst, Dieter and Van Montagu, Marc and Pedreño, María A and Martins, José and Thevelein, Johan M and Goossens, Alain},
  issn         = {0027-8424},
  journal      = {PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA},
  keywords     = {PANAX-GINSENG,TRITERPENE CYCLIZATION,GINSENOSIDE BIOSYNTHESIS,BETA-AMYRIN SYNTHASE,triterpenoid,metabolic engineering,amyrin,cyclodextrin,MEDICAGO-TRUNCATULA,CYTOCHROME-P450,CYCLODEXTRINS,GLYCYRRHIZIN,LICORICE,PLANTS},
  language     = {eng},
  number       = {4},
  pages        = {1634--1639},
  title        = {Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum},
  url          = {http://dx.doi.org/10.1073/pnas.1323369111},
  volume       = {111},
  year         = {2014},
}

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