Dietary non-starch polysaccharides impair immunity to enteric nematode infection.

BACKGROUND: The influence of diet on immune function and resistance to enteric infection and disease is becoming ever more established. Highly processed, refined diets can lead to inflammation and gut microbiome dysbiosis, whilst health-promoting dietary components such as phytonutrients and fermentable fibres are thought to promote a healthy microbiome and balanced mucosal immunity. Chicory (Cichorium intybus) is a leafy green vegetable rich in fibres and bioactive compounds that may promote gut health. RESULTS: Unexpectedly, we here show that incorporation of chicory into semisynthetic AIN93G diets renders mice susceptible to infection with enteric helminths. Mice fed a high level of chicory leaves (10% dry matter) had a more diverse gut microbiota, but a diminished type-2 immune response to infection with the intestinal roundworm Heligmosomoides polygyrus. Furthermore, the chicory-supplemented diet significantly increased burdens of the caecum-dwelling whipworm Trichuris muris, concomitant with a highly skewed type-1 immune environment in caecal tissue. The chicory-supplemented diet was rich in non-starch polysaccharides, particularly uronic acids (the monomeric constituents of pectin). In accordance, mice fed pectin-supplemented AIN93G diets had higher T. muris burdens and reduced IgE production and expression of genes involved in type-2 immunity. Importantly, treatment of pectin-fed mice with exogenous IL-25 restored type-2 responses and was sufficient to allow T. muris expulsion. CONCLUSIONS: Collectively, our data suggest that increasing levels of fermentable, non-starch polysaccharides in refined diets compromises immunity to helminth infection in mice. This diet-infection interaction may inform new strategies for manipulating the gut environment to promote resistance to enteric parasites.

Anti-Inflammatory Activity of Bryophytes Extracts in LPS-Stimulated RAW264.7 Murine Macrophages.

Bryophytes produce rare and bioactive compounds with a broad range of therapeutic potential, and many species are reported in ethnomedicinal uses. However, only a few studies have investigated their potential as natural anti-inflammatory drug candidate compounds. The present study investigates the anti-inflammatory effects of thirty-two species of bryophytes, including mosses and liverworts, on Raw 264.7 murine macrophages stimulated with lipopolysaccharide (LPS) or recombinant human peroxiredoxin (hPrx1). The 70% ethanol extracts of bryophytes were screened for their potential to reduce the production of nitric oxide (NO), an important pro-inflammatory mediator. Among the analyzed extracts, two moss species significantly inhibited LPS-induced NO production without cytotoxic effects. The bioactive extracts of Dicranum majus and Thuidium delicatulum inhibited NO production in a concentration-dependent manner with IC50 values of 1.04 and 1.54 µg/mL, respectively. The crude 70% ethanol and ethyl acetate extracts were then partitioned with different solvents in increasing order of polarity (n-hexane, diethyl ether, chloroform, ethyl acetate, and n-butanol). The fractions were screened for their inhibitory effects on NO production stimulated with LPS at 1 ng/mL or 10 ng/mL. The NO production levels were significantly affected by the fractions of decreasing polarity such as n-hexane and diethyl ether ones. Therefore, the potential of these extracts to inhibit the LPS-induced NO pathway suggests their effective properties in attenuating inflammation and could represent a perspective for the development of innovative therapeutic agents.

Effects of extraction parameters on lipid profiling of mosses using UPLC-ESI-QTOF-MS and multivariate data analysis.

INTRODUCTION: Non-target lipid profiling by using ultra-performance liquid chromatography coupled to electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS) has been used extensively in the past decades in plant studies. However, the lipidomes of bryophytes have only been scarcely studied, although they are the second largest group in plant kingdom. OBJECTIVES: We evaluated the effects of different cell disruption methods (no disruption, shake, ultrasound, and bead beating), and storage conditions (air-dried, freeze-dried, and fresh frozen) of five moss species (including Racomitrium lanuginosum B and D, Philonotis fontana, Sphagnum teres, and Hylocomium splendens). METHODS: The lipid profiling results of each extraction parameter were analyzed by using multivariate data analysis including unsupervised principal component analysis and supervised orthogonal projections to latent structures discriminant analysis. RESULTS: The results showed that extraction with bead beating resulted in the highest lipid content and the most detected features, but these were caused by the contamination from plastic tubes. Minor lipid metabolite changes were found in shaking and ultrasonication methods when compared with no disruption method. Significant amounts of phosphatidylcholine, diacylglyceryltrimethylhomoserine and their lyso lipids were observed in air-dried moss tissues, whereas diacylglycerol, triacylglycerol and ceramide were mostly exclusively detected when fresh frozen tissues were used for extraction. CONCLUSION: We concluded that lipid extraction using fresh frozen samples with ultrasound assistance provide the most original lipid composition and gave a relatively high lipid content.

Exploring evolutionary theories of plant defence investment using field populations of the deadly carrot.

BACKGROUND AND AIMS: There are a number of disparate models predicting variation in plant chemical defences between species, and within a single species over space and time. These can give conflicting predictions. Here we review a number of these theories, before assessing their power to predict the spatial-temporal variation of thapsigargins between and within populations of the deadly carrot (Thapsia garganica). By utilizing multiple models simultaneously (optimum defence theory, growth rate hypothesis, growth-differentiation balance hypothesis, intra-specific framework and resource exchange model of plant defence), we will highlight gaps in their predictions and evaluate the performance of each. METHODS: Thapsigargins are potent anti-herbivore compounds that occur in limited richness across the different plant tissues of T. garganica, and therefore represent an ideal system for exploring these models. Thapsia garganica plants were collected from six locations on the island of Ibiza, Spain, and the thapsigargins quantified within reproductive, vegetative and below-ground tissues. The effects of sampling time, location, mammalian herbivory, soil nutrition and changing root-associated fungal communities on the concentrations of thapsigargins within these in situ observations were analysed, and the results were compared with our model predictions. KEY RESULTS: The models performed well in predicting the general defence strategy of T. garganica and the above-ground distribution of thapsigargins, but failed to predict the considerable proportion of defences found below ground. Models predicting variation over environmental gradients gave conflicting and less specific predictions, with intraspecific variation remaining less understood. CONCLUSION: Here we found that multiple models predicting the general defence strategy of plant species could likely be integrated into a single model, while also finding a clear need to better incorporate below-ground defences into models of plant chemical defences. We found that constitutive and induced thapsigargins differed in their regulation, and suggest that models predicting intraspecific defences should consider them separately. Finally, we suggest that in situ studies be supplemented with experiments in controlled environments to identify specific environmental parameters that regulate variation in defences within species.

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.

Insights into Heterologous Biosynthesis of Arteannuin B and Artemisinin in Physcomitrella patens.

: Metabolic engineering is an integrated bioengineering approach, which has made considerable progress in producing terpenoids in plants and fermentable hosts. Here, the full biosynthetic pathway of artemisinin, originating from Artemisia annua, was integrated into the moss Physcomitrella patens. Different combinations of the five artemisinin biosynthesis genes were ectopically expressed in P. patens to study biosynthesis pathway activity, but also to ensure survival of successful transformants. Transformation of the first pathway gene, ADS, into P. patens resulted in the accumulation of the expected metabolite, amorpha-4,11-diene, and also accumulation of a second product, arteannuin B. This demonstrates the presence of endogenous promiscuous enzyme activity, possibly cytochrome P450s, in P. patens. Introduction of three pathway genes, ADS-CYP71AV1-ADH1 or ADS-DBR2-ALDH1 both led to the accumulation of artemisinin, hinting at the presence of one or more endogenous enzymes in P. patens that can complement the partial pathways to full pathway activity. Transgenic P. patens lines containing the different gene combinations produce artemisinin in varying amounts. The pathway gene expression in the transgenic moss lines correlates well with the chemical profile of pathway products. Moreover, expression of the pathway genes resulted in lipid body formation in all transgenic moss lines, suggesting that these may have a function in sequestration of heterologous metabolites. This work thus provides novel insights into the metabolic response of P. patens and its complementation potential for A. annua artemisinin pathway genes. Identification of the related endogenous P. patens genes could contribute to a further successful metabolic engineering of artemisinin biosynthesis, as well as bioengineering of other high-value terpenoids in P. patens.

Evaluation of synthetic promoters in Physcomitrella patens.

Securing a molecular toolbox including diverse promoters is essential for genome engineering. However, native promoters have limitations such as the available number or the length of the promoter. In this work, three short synthetic promoters were characterized by using the yellow fluorescent protein Venus. All of the tested promoters were active and showed higher mRNA expression than housekeeping gene PpAct7, and similar protein expression level to the AtUBQ10 promoter. This study shows that few cis-regulatory elements are enough to establish a strong promoter for continuous expression of genes in plants. Along with this, the study enhance the number of available promotors to be used in P. patens. It also demonstrates the potential to construct multiple non-native promoters on demand, which would aid to resolve one bottleneck in multiple pathway expression in P. patens and other plants.

Localization and in-Vivo Characterization of Thapsia garganica CYP76AE2 Indicates a Role in Thapsigargin Biosynthesis.

The Mediterranean plant Thapsia garganica (dicot, Apiaceae), also known as deadly carrot, produces the highly toxic compound thapsigargin. This compound is a potent inhibitor of the sarcoplasmic-endoplasmic reticulum Ca2+-ATPase calcium pump in mammals and is of industrial importance as the active moiety of the anticancer drug mipsagargin, currently in clinical trials. Knowledge of thapsigargin in planta storage and biosynthesis has been limited. Here, we present the putative second step in thapsigargin biosynthesis, by showing that the cytochrome P450 TgCYP76AE2, transiently expressed in Nicotiana benthamiana, converts epikunzeaol into epidihydrocostunolide. Furthermore, we show that thapsigargin is likely to be stored in secretory ducts in the roots. Transcripts from TgTPS2 (epikunzeaol synthase) and TgCYP76AE2 in roots were found only in the epithelial cells lining these secretory ducts. This emphasizes the involvement of these cells in the biosynthesis of thapsigargin. This study paves the way for further studies of thapsigargin biosynthesis.

Physcomitrella patens, a versatile synthetic biology chassis.

KEY MESSAGE: During three decades the moss Physcomitrella patens has been developed to a superb green cell factory with the first commercial products on the market. In the past three decades the moss P. patens has been developed from an obscure bryophyte to a model organism in basic biology, biotechnology, and synthetic biology. Some of the key features of this system include a wide range of Omics technologies, precise genome-engineering via homologous recombination with yeast-like efficiency, a certified good-manufacturing-practice production in bioreactors, successful upscaling to 500 L wave reactors, excellent homogeneity of protein products, superb product stability from batch-to-batch, and a reliable procedure for cryopreservation of cell lines in a master cell bank. About a dozen human proteins are being produced in P. patens as potential biopharmaceuticals, some of them are not only similar to their animal-produced counterparts, but are real biobetters with superior performance. A moss-made pharmaceutical successfully passed phase 1 clinical trials, a fragrant moss, and a cosmetic moss-product is already on the market, highlighting the economic potential of this synthetic biology chassis. Here, we focus on the features of mosses as versatile cell factories for synthetic biology and their impact on metabolic engineering.

Role of the Colletotrichum acutatum sesquiterpene synthase CaTPS in the biosynthesis of sesquiterpenoids.

Colletotrichum acutatum is a major fungal pathogen of fruit crops, which causes severe yield losses in strawberry production. A potential key factor in plant-pathogen interactions is fungal sesquiterpenoids which have mycotoxic and phytotoxic activities. The first committed step in sesquiterpenoid biosynthesis is performed by sesquiterpene synthases (TPS). Only a few TPSs have been functionally characterized from filamentous fungi and none from the genus Colletotrichum. Despite being an important fungal pathogen to agriculture, it is poorly understood at the molecular and chemical levels. The terpenoid biochemistry in Coll. acutatum strain SA 0-1 was studied and one Coll. acutatum TPS (CaTPS) was successfully cloned and characterized in yeast. CaTPS catalyses the biosynthesis of multiple sesquiterpenoids. The two major products are ß-caryophyllene and an unidentified sesquiterpenoid along with α-humulene as one of the minor sesquiterpenoid products. These products were also secreted by the fungus in strawberry fruit medium along with several other sesquiterpenoids indicating other TPSs are active during in vitro growth. ß-Caryophyllene and α-humulene are known cytotoxic products important for ecological interactions and are produced by SA 0-1. Interestingly, a gene expression analysis using quantitative real-time PCR revealed a significant increase in expression of CaTPS during strawberry fruit infection, thus indicating that it could be involved in fruit infection. This is, we believe, the first characterization of TPS in Colletotrichum spp. and terpenoid profiles of Coll. acutatum, which could facilitate studies on the role of terpenoids in the ecology of Coll. acutatum.

The decision to germinate is regulated by divergent molecular networks in spores and seeds.

Dispersal is a key step in land plant life cycles, usually via formation of spores or seeds. Regulation of spore- or seed-germination allows control over the timing of transition from one generation to the next, enabling plant dispersal. A combination of environmental and genetic factors determines when seed germination occurs. Endogenous hormones mediate this decision in response to the environment. Less is known about how spore germination is controlled in earlier-evolving nonseed plants. Here, we present an in-depth analysis of the environmental and hormonal regulation of spore germination in the model bryophyte Physcomitrella patens (Aphanoregma patens). Our data suggest that the environmental signals regulating germination are conserved, but also that downstream hormone integration pathways mediating these responses in seeds were acquired after the evolution of the bryophyte lineage. Moreover, the role of abscisic acid and diterpenes (gibberellins) in germination assumed much greater importance as land plant evolution progressed. We conclude that the endogenous hormone signalling networks mediating germination in response to the environment may have evolved independently in spores and seeds. This paves the way for future research about how the mechanisms of plant dispersal on land evolved.

Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing.

The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to a huge number of different cytochromes P450s (from 50 to several hundred within one plant). Within the eudicotyledons, PORs can be divided into two major clades, POR 1 and POR 2. Based on our own sequencing analysis and publicly available data, we have identified 45 PORs from the angiosperm order Apiales. These were subjected to a phylogenetic analysis along with 237 other publicly available (NCBI and oneKP) POR sequences found within the clade Asterids. Here, we show that the order Apiales only harbor members of the POR 2 clade, which are further divided into two distinct subclades. This is in contrast to most other eudicotyledon orders that have both POR 1 and POR 2. This suggests that through gene duplications and one gene deletion, Apiales only contain members of the POR 2 clade. Three POR 2 isoforms from Thapsia garganica L., Apiaceae, were all full-length in an Illumina root transcriptome dataset (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes.

Two key polymorphisms in a newly discovered allele of the Vitis vinifera TPS24 gene are responsible for the production of the rotundone precursor α-guaiene.

Rotundone was initially identified as a grape-derived compound responsible for the peppery aroma of Shiraz wine varieties. It has subsequently been found in black and white pepper and several other spices. Because of its potent aroma, the molecular basis for rotundone formation is of particular relevance to grape and wine scientists and industry. We have identified and functionally characterized in planta a sesquiterpene synthase, VvGuaS, from developing grape berries, and have demonstrated that it produces the precursor of rotundone, α-guaiene, as its main product. The VvGuaS enzyme is a novel allele of the sesquiterpene synthase gene, VvTPS24, which has previously been reported to encode VvPNSeInt, an enzyme that produces a variety of selinene-type sesquiterpenes. This newly discovered VvTPS24 allele encodes an enzyme 99.5% identical to VvPNSeInt, with the differences comprising just 6 out of the 561 amino acid residues. Molecular modelling of the enzymes revealed that two of these residues, T414 and V530, are located in the active site of VvGuaS within 4 Å of the binding-site of the substrate, farnesyl pyrophosphate. Mutation of these two residues of VvGuaS into the corresponding polymorphisms in VvPNSeInt results in a complete functional conversion of one enzyme into the other, while mutation of each residue individually produces an intermediate change in the product profile. We have therefore demonstrated that VvGuaS, an enzyme responsible for production of the rotundone precursor, α-guaiene, is encoded by a novel allele of the previously characterized grapevine gene VvTPS24 and that two specific polymorphisms are responsible for functional differences between VvTPS24 alleles.

Anthelmintic activity of chicory (Cichorium intybus): in vitro effects on swine nematodes and relationship to sesquiterpene lactone composition.

Chicory is a perennial crop that has been investigated as a forage source for outdoor-reared ruminants and pigs, and has been reported to have anthelmintic properties. Here, we investigated in vitro anthelmintic effects of forage chicory-extracts against the highly prevalent swine parasites Ascaris suum and Oesophagostomum dentatum. Methanol extracts were prepared and purified from two different cultivars of chicory (Spadona and Puna II). Marked differences were observed between the anthelmintic activity of extracts from the two cultivars. Spadona extracts had potent activity against A. suum third (L3) and fourth (L4) - stage larvae, as well as O. dentatum L4 and adults, whereas Puna II extracts had less activity against A. suum and no activity towards O. dentatum L4. Transmission-electron microscopy of A. suum L4 exposed to Spadona extracts revealed only subtle changes, perhaps indicative of a specific anthelmintic effect rather than generalized toxicity. Ultra-high liquid chromatography-mass spectrometry analysis revealed that the purified extracts were rich in sesquiterpene lactones (SL), and that the SL profile differed significantly between cultivars. This is the first report of anthelmintic activity of forage chicory towards swine nematodes. Our results indicate a significant anthelmintic effect, which may possibly be related to SL composition.

Evolution of substrate recognition sites (SRSs) in cytochromes P450 from Apiaceae exemplified by the CYP71AJ subfamily.

BACKGROUND: Large proliferations of cytochrome P450 encoding genes resulting from gene duplications can be termed as 'blooms', providing genetic material for the genesis and evolution of biosynthetic pathways. Furanocoumarins are allelochemicals produced by many of the species in Apiaceaous plants belonging to the Apioideae subfamily of Apiaceae and have been described as being involved in the defence reaction against phytophageous insects. RESULTS: A bloom in the cytochromes P450 CYP71AJ subfamily has been identified, showing at least 2 clades and 6 subclades within the CYP71AJ subfamily. Two of the subclades were functionally assigned to the biosynthesis of furanocoumarins. Six substrate recognition sites (SRS1-6) important for the enzymatic conversion were investigated in the described cytochromes P450 and display significant variability within the CYP71AJ subfamily. Homology models underline a significant modification of the accession to the iron atom, which might explain the difference of the substrate specificity between the cytochromes P450 restricted to furanocoumarins as substrates and the orphan CYP71AJ. CONCLUSION: Two subclades functionally assigned to the biosynthesis of furanocoumarins and four other subclades were identified and shown to be part of two distinct clades within the CYP71AJ subfamily. The subclades show significant variability within their substrate recognition sites between the clades, suggesting different biochemical functions and providing insights into the evolution of cytochrome P450 'blooms' in response to environmental pressures.

Thapsigargin--from Thapsia L. to mipsagargin.

The sesquiterpene lactone thapsigargin is found in the plant Thapsia garganica L., and is one of the major constituents of the roots and fruits of this Mediterranean species. In 1978, the first pharmacological effects of thapsigargin were established and the full structure was elucidated in 1985. Shortly after, the overall mechanism of the Sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) inhibition that leads to apoptosis was discovered. Thapsigargin has a potent antagonistic effect on the SERCA and is widely used to study Ca2+-signaling. The effect on SERCA has also been utilized in the treatment of solid tumors. A prodrug has been designed to target the blood vessels of cancer cells; the death of these blood vessels then leads to tumor necrosis. The first clinical trials of this drug were initiated in 2008, and the potent drug is expected to enter the market in the near future under the generic name Mipsagargin (G-202). This review will describe the discovery of the new drug, the on-going elucidation of the biosynthesis of thapsigargin in the plant and attempts to supply the global market with a novel potent anti-cancer drug.

Tamariscol biosynthesis in Frullania tamarisci.

The liverwort Frullania tamarisci (L.) Dumort produces large amounts of terpenoids, among others the sesquiterpene alcohol tamariscol. Tamariscol has an earthy woody fragrance, and the use in perfurmes and production of it was patented in 1984. The microbial terpene synthase-like (MTPSL) enzyme FtMTPSL6 is shown to be responsible for the biosynthesis of tamariscol. FtMTPSL6 was obtained through RNA sequencing of wild growing F. tamarisci along with six other MTPSLs (FtMTPSL1-7). The biochemical activity was determined for three of them, and two others where metabolic active, but the product could not be identified. The three characterized enzymes are the tamariscol synthase (FtMTPSL6), copaene synthase (FtMTPSL1) and gurjunene synthase (FtMTPSL3). Thus, the biosynthesis of the economically attractive compound tamariscol is established, and this opens up for further exploitation of this molecule.

The telomere-to-telomere (T2T) genome of Peucedanum praeruptorum Dunn provides insights into the genome evolution and coumarin biosynthesis.

BACKGROUND: Traditional Chinese medicine has used Peucedanum praeruptorum Dunn (Apiaceae) for a long time. Various coumarins, including the significant constituents praeruptorin (A-E), are the active constituents in the dried roots of P. praeruptorum. Previous transcriptomic and metabolomic studies have attempted to elucidate the distribution and biosynthetic network of these medicinal-valuable compounds. However, the lack of a high-quality reference genome impedes an in-depth understanding of genetic traits and thus the development of better breeding strategies. RESULTS: A telomere-to-telomere (T2T) genome was assembled for P. praeruptorum by combining PacBio HiFi, ONT ultra-long, and Hi-C data. The final genome assembly was approximately 1.798 Gb, assigned to 11 chromosomes with genome completeness >98%. Comparative genomic analysis suggested that P. praeruptorum experienced 2 whole-genome duplication events. By the transcriptomic and metabolomic analysis of the coumarin metabolic pathway, we presented coumarins' spatial and temporal distribution and the expression patterns of critical genes for its biosynthesis. Notably, the COSY and cytochrome P450 genes showed tandem duplications on several chromosomes, which may be responsible for the high accumulation of coumarins. CONCLUSIONS: A T2T genome for P. praeruptorum was obtained, providing molecular insights into the chromosomal distribution of the coumarin biosynthetic genes. This high-quality genome is an essential resource for designing engineering strategies for improving the production of these valuable compounds.

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.

Transcriptome analysis of Thapsia laciniata Rouy provides insights into terpenoid biosynthesis and diversity in Apiaceae.

Thapsia laciniata Rouy (Apiaceae) produces irregular and regular sesquiterpenoids with thapsane and guaiene carbon skeletons, as found in other Apiaceae species. A transcriptomic analysis utilizing Illumina next-generation sequencing enabled the identification of novel genes involved in the biosynthesis of terpenoids in Thapsia. From 66.78 million HQ paired-end reads obtained from T. laciniata roots, 64.58 million were assembled into 76,565 contigs (N50: 1261 bp). Seventeen contigs were annotated as terpene synthases and five of these were predicted to be sesquiterpene synthases. Of the 67 contigs annotated as cytochromes P450, 18 of these are part of the CYP71 clade that primarily performs hydroxylations of specialized metabolites. Three contigs annotated as aldehyde dehydrogenases grouped phylogenetically with the characterized ALDH1 from Artemisia annua and three contigs annotated as alcohol dehydrogenases grouped with the recently described ADH1 from A. annua. ALDH1 and ADH1 were characterized as part of the artemisinin biosynthesis. We have produced a comprehensive EST dataset for T. laciniata roots, which contains a large sample of the T. laciniata transcriptome. These transcriptome data provide the foundation for future research into the molecular basis for terpenoid biosynthesis in Thapsia and on the evolution of terpenoids in Apiaceae.