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

Generation of homoplastomic transplastomic plants expressing Cr-PTOX1.

<p>Schematic representation of the plastome region of the wildtype (A) and the transplastomic plant line Cr-PTOX1-I (B) analyzed by Southern blot analysis. Cr-PTOX1-I sequences were cloned in vector pHK40 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041722#pone.0041722-Kuroda1" target="_blank">[22]</a>. The positions of the restriction enzyme BglII used to digest the genomic DNA are shown. The dotted lines represent the size of the expected fragments to be released from wildtype as well as Cr-PTOX1-I after restriction. (C) Total genomic DNA from WT as well as Cr-PTOX1-I was digested and hybridized with rrn16/rps12 digoxigenin labelled probe amplified from WT. (D) Maternal inheritance assay of Cr-PTOX1-I transplastomic plant line. Seeds of Cr-PTOX1-I and WT plants were grown on MS plates with or without 500 mg L⁻¹ spectinomycin at room temperature. Abbreviations: P<i>rrn</i> = 16SRNA operon promoter, T<i>rbcL</i> = Rubisco large subunit terminator, P<i>psbA</i> = <i>psbA</i> promoter, T<i>psbA</i> = <i>psbA</i> terminator, WT = wild type, T = transplastomic, ptDNA = Plastid DNA.</p

Immunodetection of Cr-PTOX1 protein by BN and 2D SDS-PAGE.

<p>Thylakoid membranes from Cr-PTOXI-I or WT containing 4 µg chlorophyll were solubilised and separated by BN-PAGE. One of the BN-PAGE gel lanes was stained with Coomassie blue (A) and others were run in the second dimension by denaturing SDS-PAGE. One of the gels obtained was silver-stained (B) while other two were immunoblotted with anti-HA tag (C) or anti-D1 antibodies (D).</p

Multiple sequence alignment and phylogenetic analysis of PTOX.

<p>(A) Neighbour-joining phylogenetic dendrogram (spider) based upon an alignment of complete amino acid sequences of PTOX molecules. Grouping in different shades based upon structural and functional homology of PTOX polypeptides found in different species. Numbers at nodes indicate bootstrap confidence values (1000 replicates). The PTOX amino acid sequences used were from <i>Arabidopsis thaliana</i> GenBank accession number: CAA06190, tomato (<i>Lycopersicon esculentum</i>) GenBank accession number: AAG02286, rice (<i>Oryza sativa</i>; cultivar <i>japonica</i>) NCBI accession number: NP_001054199, wheat (<i>Triticum aestivum</i>) GenBank accession number: AAG00450, maize (<i>Zea mays</i>) NCBI accession number: NP_001150780, coffee (<i>Coffea canephora</i>) GenBank accession number: ABB70513, pepper (<i>Capsicum annuum</i>) GenBank accession number: AAG02288, <i>Guillardia theta</i> GenBank accession number: CAI77910, <i>Chlamydomonas reinhardtii</i> NCBI accession number: AF494290 (PTOX1) and NCBI accession number: XP_001703466 (PTOX2), <i>Ostreococcus tauri</i> (green alga) GenBank accession number: CAL58090, <i>Bigelowiella natans</i> (chlorarachniophytes) GenBank accession number: AAP79178, <i>Anabaena variabilis</i> ATCC 29413 (cyanobacteria) GenBank accession number: ABA21297, Nostoc sp. PCC 7120 (cyanobacteria) NCBI NP_486136, <i>Prochlorococcus marinus</i> subsp. Pastoris CCMP1986 (cyanobacteria) NCBI accession number: NP_892455, <i>Prochlorococcus marinus</i> Subsp. ASNC729 (cyanobacteria) Genbank accession number: ABE11017, <i>Prochlorococcus marinus</i> str. MIT 9312 (cyanobacteria) NCBI accession number: YP_396838, <i>Prochlorococcus marinus</i> str. NATL2A (cyanobacteria) NCBI accession number: YP_291624, <i>Synechococcus</i> sp. WH 8102 (cyanobacteria) NCBI accession number: NP_896980, <i>Synechococcus</i> sp. CC9902 (cyanobacteria) NCBI accession number: YP_376451, and <i>Synechococcus</i> sp. BL107 (cyanobacteria) NCBI: ZP_01468216. (B) Multiple sequence alignment of PTOX from <i>Chlamydomonas</i> and <i>Arabidopsis</i>. Conserved iron-binding residues are indicated by black arrows, whereas, exon 8 is boxed. Conserved sequences are shaded black. The transit peptide for At-PTOX is underlined.</p

Detection of Cr-PTOX1 by SDS-PAGE and Western blot analysis.

<p>Total proteins equivalent to 1 µg of chlorophyll were loaded per well from PTOX1-I plant leaves grown in a greenhouse at high light (125 µmol photons m⁻² s⁻¹) and analysed either by running on a 15% (w/v) denaturing polyacrylamide gel and stained by Coomassie Blue (A) or transferring to PVDF for immunodetection carried out using an anti-HA tag antibody (B). Protein samples from <i>Chlamydomonas reinhardtii</i> expressing HA-tagged Light Harvesting Complex b (HA-LHC b) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041722#pone.0041722-Mussgnug1" target="_blank">[49]</a> were used as a positive immunoblotting control. (C) Tobacco plants were grown in a growth room at 50 µmol photons m⁻² s⁻¹. 5 µg of thylakoids (based on chlorophyll) from tobacco, wild type (WT) as well Cr-PTOX1 expressing plants and <i>C. reinhardtii</i> (wild type) were loaded per well for SDS-PAGE and immunoblotting, which was carried out using an anti-PTOX1 antibody. Upper panel shows a Coomassie-stained gel, whereas, the bottom panel shows the immunoblot. (D) Differential extraction of thylakoids to determine whether Cr-PTOX1 is being targeted to the membrane. Thylakoids extracted from Cr-PTOX1-I plant leaves were washed with different buffers and centrifuged. The supernatant and pellet fractions (∼1 µg chlorophyll per well) were loaded and immunoblotted using different antibodies against membrane bound proteins.</p

Production of leafy biomass using temporary immersion bioreactors: an alternative platform to express proteins in transplastomic plants with drastic phenotypes.

Chloroplast transformation technology is a promising approach for the production of foreign proteins in plants with expression levels of up to 70 % of total soluble protein (TSP) achieved in tobacco. However, expression of foreign protein in the chloroplast can lead to drastic or even lethal effects in transplastomic plants grown in soil, thereby potentially limiting the applicability of this technology. For instance, previous attempts to express the outer surface protein A (OspA) from Borrelia burgdorferi in tobacco chloroplasts led to plant death when expressed at 10 % TSP. We show here that this earlier transplastomic line, as well as a new plant line, OspA:YFP, expressing OspA fused to the yellow fluorescent protein, can be propagated in temporary immersion bioreactors (TIBs) using AlkaBurst™ technology to produce leafy biomass that expressed OspA at levels of up to 7.6 % TSP, to give a maximum yield of OspA of about 108 mg/L. Our results show that TIBs provide an alternative method for the production of transplastomic biomass expressing proteins toxic for plants and is a particularly useful approach when 'absolute' containment is required

Involvement of Cr-PTOX1 in PQ oxidation and effect of propyl gallate on Cr-PTOX1 activity.

<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 by measuring chlorophyll fluorescence. The fluorescence decay in leaf discs of WT (black trace) and Cr-PTOX1-I plants (red trace) were measured after 1-hour dark adaptation (A). Leaf discs of both WT (B) and Cr-PTOX1-I plants (C) were treated with (broken line) or without (unbroken line) 1 mM propyl gallate (PG) for a period of 3 hours in the dark. Fluorescence values (arbitrary units) are shown on y-axis, whereas, time is shown on x-axis.</p