Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 111, Issue 4, Pages 1634-1639Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.1323369111
Keywords
cyclodextrin; amyrin; metabolic engineering; triterpenoid
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Funding
- Agency for Innovation by Science and Technology (Strategisch Basisonderzoek Project) [SBO040093]
- European Union Seventh Framework Programme FP7/2007-2013 [222716]
- VIB International Fellowship Program
- European Cooperation in Science and Technology Action FA1006-Plant Engine for a short-term scientific mission grant
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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.
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