Anti-protozoal activity of extracts from chicory (Cichorium intybus) against Cryptosporidium parvum in cell culture.

Cryptosporidium spp. are responsible for severe public health problems and livestock production losses. Treatment options are limited to only one drug available for human and bovine cryptosporidiosis, respectively, and both drugs exhibit only partial efficacy. Sesquiterpene lactones (SL) are plant bioactive compounds that function as a defence mechanism against herbivores. SL have demonstrated anti-parasitic properties against a range of parasitic taxa but knowledge about their anti-Cryptosporidium efficacy is limited. The effect of SL-rich leaf and root extracts from chicory (Cichorium intybus cv. Spadona) was investigated using human colon adenocarcinoma (HCT-8) cells infected with Cryptosporidium parvum. C. parvum oocysts were inoculated onto the cell monolayer and i) incubated for 4 hours with extracts (leaf and root extracts 300, 150, 75, 37.5, 18.75 and 9.375 µg/mL) in triplicates followed by incubation in bioactive free media (sporozoite invasion assays) or ii) incubated for 4 hours in bioactive free media followed by 48-hours incubation with extracts (growth inhibition assays). Extract toxicity on HCT-8 cells was assessed via water-soluble tetrazolium (WST)-1 assay prior to quantifying parasitic growth via immunofluorescence. Both extracts demonstrated dose-dependent inhibition in the growth inhibition assays (p = < 0.0001 for both extracts) but not in the invasion assays. Anti-parasitic activity did not appear to be solely related to SL content, with the extract with lower SL content (leaf) exhibiting higher inhibition at 300 µg/ml. However, given the limited treatment options available for Cryptosporidium spp., our study encourages further investigation into the use of chicory extracts to identify novel active compound(s) inhibiting these protozoa.

In planta and in silico characterization of five sesquiterpene synthases from Vitis vinifera (cv. Shiraz) berries.

MAIN CONCLUSION: Five Vitis vinifera sesquiterpene synthases were characterized, two was previously uncharacterized, one being a caryophyllene/cubebene synthase and the other a cadinene synthase. Residue differences with other Vitis sesquiterpene synthases are described. The biochemical composition of grape berries at harvest can have a profound effect on the varietal character of the wine produced. Sesquiterpenes are an important class of volatile compounds produced in grapes that contribute to the flavor and aroma of wine, making the elucidation of their biosynthetic origin an important field of research. Five cDNAs corresponding to sesquiterpene synthase genes (TPSs) were isolated from Shiraz berries and expressed in planta in Nicotiana benthamiana followed by chemical characterization by GC-MS. Three of the TPS cDNAs were isolated from immature berries and two were isolated from ripe Shiraz berries. Two of the investigated enzymes, TPS26 and TPS27, have been previously investigated by expression in E. coli, and the in planta products generally correspond to these previous studies. The enzyme TPS07 differed by eight amino acids (none of which are in the active site) from germacrene B and D synthase isolated from Gewürztraminer grapes and characterized in vitro. Here in planta characterization of VvShirazTPS07 yielded ylangene, germacrene D and several minor products. Two of the enzymes isolated from immature berries were previously uncharacterized enzymes. VvShirazTPS-Y1 produced cadinene as a major product and at least 17 minor sesquiterpenoid skeletons. The second, VvShirazTPS-Y2, was characterized as a caryophyllene/cubebene synthase, a combination of products not previously reported from a single enzyme. Using in silico methods, we identified residues that could play key roles regarding differences in product formation of these enzymes. The first ring closure that is either a 1,10- or 1,11-ring closure is likely controlled by three neighboring amino acids in helices G1, H2, and J. As for many other investigated TPS enzymes, we also observe that only a few residues can account for radical changes in product formation.

Stable heterologous expression of biologically active terpenoids in green plant cells.

Plants biosynthesize a great diversity of biologically active small molecules of interest for fragrances, flavors, and pharmaceuticals. Among specialized metabolites, terpenoids represent the greatest molecular diversity. Many terpenoids are very complex, and total chemical synthesis often requires many steps and difficult chemical reactions, resulting in a low final yield or incorrect stereochemistry. Several drug candidates with terpene skeletons are difficult to obtain by chemical synthesis due to their large number of chiral centers. Thus, biological production remains the preferred method for industrial production for many of these compounds. However, because these chemicals are often found in low abundance in the native plant, or are produced in plants which are difficult to cultivate, there is great interest in engineering increased production or expression of the biosynthetic pathways in heterologous hosts. Although there are many examples of successful engineering of microbes such as yeast or bacteria to produce these compounds, this often requires extensive changes to the host organism's metabolism. Optimization of plant gene expression, post-translational protein modifications, subcellular localization, and other factors often present challenges. To address the future demand for natural products used as drugs, new platforms are being established that are better suited for heterologous production of plant metabolites. Specifically, direct metabolic engineering of plants can provide effective heterologous expression for production of valuable plant-derived natural products. In this review, our primary focus is on small terpenoids and we discuss the benefits of plant expression platforms and provide several successful examples of stable production of small terpenoids in plants.

Identification and characterization of a kunzeaol synthase from Thapsia garganica: implications for the biosynthesis of the pharmaceutical thapsigargin.

Thapsigargin is a major terpenoid constituent of Thapsia garganica root. Owing to its potent antagonistic effect on the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, thapsigargin has been widely used to study Ca2+ signalling and is also a potential drug for prostate cancer. Despite its importance, thapsigargin biosynthesis in T. garganica remains unknown. In order to decipher thapsigargin biosynthesis, deep transcript sequencing (454 and Illumina) of the T. garganica root was performed, and two terpene synthases (TgTPS1/2) were identified. Functional characterization of their encoded enzymes in a metabolically engineered yeast revealed that TgTPS1 synthesized δ-cadinene, whereas TgTPS2 produced ten distinct terpenoids. However, cultivation of the TgTPS2-expressing yeast in pH-maintained conditions (pH 6-7) yielded one major oxygenated sesquiterpenoid, suggesting that formation of multiple terpenoids was caused by acidity. The major terpene product from TgTPS2 was identified as 6ß-hydroxygermacra-1(10),4-diene (kunzeaol) by mass-fragmentation pattern, retention index, the nature of its acid-induced degradation and NMR. Also, recombinant TgTPS2 efficiently catalysed the synthesis of kunzeaol in vitro from farnesyl diphosphate with a Km of 2.6 µM and a kcat of 0.03 s-1. The present paper is the first report of a kunzeaol synthase, and a mechanism for the transformation of kunzeaol into the thapsigargin backbone is proposed.