Use of a temporary immersion bioreactor system for the sustainable production of thapsigargin in shoot cultures of Thapsia garganica

Carmen Quiñonero López; Patricia Corral; Bénédicte Lorrain-Lorrette; Karen Martinez-Swatson; Franck Michoux; Henrik Toft Simonsen

doi:10.1186/s13007-018-0346-z

Use of a temporary immersion bioreactor system for the sustainable production of thapsigargin in shoot cultures of Thapsia garganica

Plant Methods. 2018 Sep 8:14:79. doi: 10.1186/s13007-018-0346-z. eCollection 2018.

Authors

Carmen Quiñonero López^{1

2}, Patricia Corral³, Bénédicte Lorrain-Lorrette³, Karen Martinez-Swatson⁴, Franck Michoux³, Henrik Toft Simonsen¹

Affiliations

¹ 1Department of Biotechnology and Biomedicine, Faculty of Bioengineering, Technical University of Denmark, Lyngby, Denmark.
² 2Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Copenhagen, Denmark.
³ Evonik Advanced Botanicals SAS, Parçay Meslay, France.
⁴ 4Natural History Museum of Denmark, University of Copenhagen, Copenhagen, Denmark.

Abstract

Background: Thapsigargin and nortrilobolide are sesquiterpene lactones found in the Mediterranean plant Thapsia garganica L. Thapsigargin is a potent inhibitor of the sarco/endoplasmic reticulum calcium ATPase pump, inducing apoptosis in mammalian cells. This mechanism has been used to develop a thapsigargin-based cancer drug first by GenSpera and later Inspyr Therapeutics (Westlake Village, California). However, a stable production of thapsigargin is not established.

Results: In vitro regeneration from leaf explants, shoot multiplication and rooting of T. garganica was obtained along with the production of thapsigargins in temporary immersion bioreactors (TIBs). Thapsigargin production was enhanced using reduced nutrient supply in combination with methyl jasmonate elicitation treatments. Shoots grown in vitro were able to produce 0.34% and 2.1% dry weight of thapsigargin and nortrilobolide, respectively, while leaves and stems of wild T. garganica plants contain only between 0.1 and 0.5% of thapsigargin and below detectable levels of nortrilobolide. In addition, a real-time reverse transcription PCR (qRT-PCR) study was performed to study the regulatory role of the biosynthetic genes HMG-CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS), epikunzeaol synthase (TgTPS2) and the cytochrome P450 (TgCYP76AE2) of stem, leaf and callus tissues. Nadi staining showed that the thapsigargins are located in secretory ducts within these tissues.

Conclusions: Shoot regeneration, rooting and biomass growth from leaf explants of T. garganica were achieved, together with a high yield in vitro production of thapsigargin in TIBs.

Keywords: In vitro shoot cultures; Nortrilobolide; Sesquiterpenes; Temporary immersion bioreactor; TgTPS2 and TgCYP76AE2; Thapsia garganica; Thapsigargin.