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

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

Developing new strategies for the production of foreign proteins in higher plant chloroplasts

Transformation of the chloroplast genome is a technique that allows for the production of recombinant proteins in plants. Chloroplast transformation allows the very high expression of a transgene while limiting the gene flow to other species. In addition, multiple genes can be transferred in one transformation event and no gene silencing as been shown so far. Despite these advantages, little work has been directed at assessing the commercial feasibility of using the chloroplast as a means of expressing high-value proteins. In this thesis, a new expression system was developed to allow the very high expression of transgenes in the chloroplast under contained conditions. Tobacco plants were transformed with a tobacco chloroplast vector expressing green fluorescent \ protein. Cell suspensions were induced from these leaves. Using a bioreactor, I was able to demonstrate that coupling temporary immersion with a hormonal shift triggered the rapid production of plant tissue whilst retaining a high level of expression of green fluorescent protein. Another aspect of this thesis was to assess the potential of several fusion tags to improve the solubility and purification of various target proteins in transformed tobacco chloroplasts. The main proteins studied were a mannanase from coffee, which is involved in the detergent, pulp and, more recently, the bioethanol industry, as well as alpha defensin 1 peptide, which has potential therapeutic value in the treatment of several diseases such as HIV and Herpes. N- and C-terminal constructions were created with oleosin, dehydrin, fibrillin, maltose-binding protein and glutathione-S-transferase as tags. Constructs were first evaluated in Escherichia coli before being integrated into the tobacco plastome. Apart from oleo sin, all fusion proteins were successfully expressed in transplastomic tobacco. My work has identified dehydrin, GST and MBP as promising affinity-tags to be used in chloroplast transformation experiments. Finally, I describe the development of experimental tools and procedures for the transformation of the chloroplast genome of coffee, which is one of the world's major cash crops. For this, coffee-specific vectors were created and direct somatic embryogenesis employed to propagate transformed tissue.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Triterpene messages from the EU-FP7 Project TriForC

TriForC is an innovative EU-funded collaborative project that has established an integrative pipeline for the exploitation of plant triterpenes for commercialization in agriculture and pharmacology. We discuss the main outcomes of TriForC and reflect on its potential long-term impact and on the importance of EU projects for science, industry, and society

Pneumatic hydrodynamics influence transplastomic protein yields and biological responses during in vitro shoot regeneration of Nicotiana tabacum callus: Implications for bioprocess routes to plant-made biopharmaceuticals

AbstractTransplastomic plants are capable of high-yield production of recombinant biopharmaceutical proteins. Plant tissue culture combines advantages of agricultural cultivation with the bioprocess consistency associated with suspension culture. Overexpression of recombinant proteins through regeneration of transplastomic Nicotiana tabacum shoots from callus tissue in RITA® temporary immersion bioreactors has been previously demonstrated. In this study we investigated the hydrodynamics of periodic pneumatic suspension of liquid medium during temporary immersion culture (4min aeration every 8h), and the impact on biological responses and transplastomic expression of fragment C of tetanus toxin (TetC). Biomass was grown under a range of aeration rates for 3, 20 and 40-day durations. Growth, mitochondrial activity (a viability indicator) and TetC protein yields were correlated against the hydrodynamic parameters, shear rate and energy dissipation rate (perkg of medium). A critical aeration rate of 440mlmin−1 was identified, corresponding to a shear rate of 96.7s−1, pneumatic power input of 8.8mWkg−1 and initial 20-day pneumatic energy dissipation of 127Jkg−1, at which significant reductions in biomass accumulation and mitochondrial activity were observed. There was an exponential decline in TetC yields with increasing aeration rates at 40days, across the entire range of conditions tested. These observations have important implications for the optimisation and scale-up of transplastomic plant tissue culture bioprocesses for biopharmaceutical production

Changes in post-illumination Fo rise at various time points during fluorescence induction.

<p>WT and Cr-PTOX1-I plants were grown at low light (50 µmol photons m⁻² s⁻¹). 10-week-old plants were then analysed for post-illumination Fo rise. Dark-adapted WT (A) and Cr-PTOX1-I (B) leaves were illuminated by 96-µmol photons m⁻² s⁻¹ white light (AL) for various time points over a period of 30 min. Post-illumination fluorescence kinetics was monitored by placing leaves in the dark for 5 min after illuminating leaves for 5, 10, 15, 20 and 25 min. Saturating flashes of 1000 µmol photons m⁻² s⁻¹, 600 ms duration each at 1 min interval were applied throughout the period (24 measurements). Abbreviations: AL = Actinic light, MB = Measuring Beam, Fm = Maximum fluorescence from dark-adapted leaves after saturating flash, Fo = minimum fluorescence in dark-adapted leaves before illumination.</p