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.

Integrating genomic and multiomic data for Angelica sinensis provides insights into the evolution and biosynthesis of pharmaceutically bioactive compounds.

Angelica sinensis roots (Angelica roots) are rich in many bioactive compounds, including phthalides, coumarins, lignans, and terpenoids. However, the molecular bases for their biosynthesis are still poorly understood. Here, an improved chromosome-scale genome for A. sinensis var. Qinggui1 is reported, with a size of 2.16 Gb, contig N50 of 4.96 Mb and scaffold N50 of 198.27 Mb, covering 99.8% of the estimated genome. Additionally, by integrating genome sequencing, metabolomic profiling, and transcriptome analysis of normally growing and early-flowering Angelica roots that exhibit dramatically different metabolite profiles, the pathways and critical metabolic genes for the biosynthesis of these major bioactive components in Angelica roots have been deciphered. Multiomic analyses have also revealed the evolution and regulation of key metabolic genes for the biosynthesis of pharmaceutically bioactive components; in particular, TPSs for terpenoid volatiles, ACCs for malonyl CoA, PKSs for phthalide, and PTs for coumarin biosynthesis were expanded in the A. sinensis genome. These findings provide new insights into the biosynthesis of pharmaceutically important compounds in Angelica roots for exploration of synthetic biology and genetic improvement of herbal quality.

Heterologous Production of Artemisinin in Physcomitrium patens by Direct in vivo Assembly of Multiple DNA Fragments.

The sesquiterpene lactone compound artemisinin is a natural medicinal product of commercial importance. This Artemisia annua-derived secondary metabolite is well known for its antimalarial activity and has been studied in several other biological assays. However, the major shortcoming in its production and commercialization is its low accumulation in the native plant. Moreover, the chemical synthesis of artemisinin is difficult and expensive due to its complex structure. Hence, an alternative and sustainable production system of artemisinin in a heterologous host is required. Previously, heterologous production of artemisinin was achieved by Agrobacterium-mediated transformation. However, this requires extensive bioengineering of modified Nicotiana plants. Recently, a technique involving direct in vivo assembly of multiple DNA fragments in the moss, P. patens, has been successfully established. We utilized this technique to engineer artemisinin biosynthetic pathway genes into the moss, and artemisinin was obtained without further modifications with high initial production. Here, we provide protocols for establishing moss culture accumulating artemisinin, including culture preparation, transformation method, and compound detection via HS-SPME, UPLC-MRM-MS, and LC-QTOF-MS. The bioengineering of moss opens up a more sustainable, cost effective, and scalable platform not only in artemisinin production but also other high-value specialized metabolites in the future.

Overexpression of Physcomitrium patens cell cycle regulators leads to larger gametophytes.

Regulation of cell division is crucial for the development of multicellular organisms, and in plants, this is in part regulated by the D-type cyclins (CYCD) and cyclin-dependent kinase A (CDKA) complex. Cell division regulation in Physcomitrium differs from other plants, by having cell division checks at both the G1 to S and G2 to M transition, controlled by the CYCD1/CDKA2 and CYCD2/CDKA1 complexes, respectively. This led us to hypothesize that upregulation of cell division could be archived in Bryophytes, without the devastating phenotypes observed in Arabidopsis. Overexpressing lines of PpCYCD1, PpCYCD2, PpCDKA1, or PpCDKA2 under Ubiquitin promotor control provided transcriptomic and phenotypical data that confirmed their involvement in the G1 to S or G2 to M transition control. Interestingly, combinatorial overexpression of all four genes produced plants with dominant PpCDKA2 and PpCYCD1 phenotypes and led to plants with twice as large gametophores. No detrimental phenotypes were observed in this line and two of the major carbon sinks in plants, the cell wall and starch, were unaffected by the increased growth rate. These results show that the cell cycle characteristics of P. patens can be manipulated by the ectopic expression of cell cycle regulators.

Lipidomes of Icelandic bryophytes and screening of high contents of polyunsaturated fatty acids by using lipidomics approach.

Bryophytes (mosses, liverworts, and hornworts) have interested researchers because of their high chemical diversity and their potential uses in pharmaceutical, food, and cosmetic industries. Specifically, long-chain polyunsaturated fatty acids (l-PUFA) such as arachidonic acid (AA) and eicosapentaenoic acid (EPA) are commonly found in bryophytes, but not in vascular plants. Bryophytes accumulate PUFAs in cold or even freezing temperature to keep the cell fluidity. Iceland has a long history of bryophyte vegetation. These bryophytes are highly adapted to the harsh environment in Iceland and therefore are expected to produce high amounts of PUFAs. However, despite the fact that hundreds of mosses and liverworts have been found in Iceland, their lipid profiles largely remain unknown. In this study, we performed untargeted lipidomics by using UPLC-ESI-QTOF-MS as a rapid screening strategy to examine the lipid compositions of 39 local bryophyte species in Iceland and aimed to find high AA and EPA producers. A total of 280 lipid molecular species from 15 lipid classes were quantified with isotope-labeled internal standards. AA and EPA were abundantly distributed in the phospholipids (mainly PC and PE) and glycerolipids (MGDG and DGDG) in six moss species, namely Racomotrium lanuginosum, R. ericoides, Bryum psedotriquetrium, Plagiomnium ellipticum, Hylocomium splendens, and Rhytidiadelphus triquetrus. Two of the six species (B. psedotriquetrium and H. splendens) also accumulated high concentrations of PUFA-containing-triacylglycerols.

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.

Lipidomic analysis of moss species Bryum pseudotriquetrum and Physcomitrium patens under cold stress.

Bryophytes, which lack lignin for protection, support themselves in harsh environments by producing various chemicals. In response to cold stress, lipids play a crucial role in cell adaptation and energy storage. Specifically, bryophytes survive at low temperatures by producing very long-chain polyunsaturated fatty acids (vl-PUFAs). The in-depth understanding of the lipid response to cold stress of bryophytes was studied by performing lipid profiling using ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry (UHPLC-QTOF-MS). Two moss species (Bryum pseudotriquetrum and Physcomitrium patens) cultivated at 23°C and at 10°C were included in this study. Relative quantitative lipid concentrations were compared and the potential lipid biomarkers were identified by multivariate statistical analysis in each species. In B. pseudotriquetrum, it was observed that the phospholipids and glycolipids increased under cold stress, while storage lipids decreased. The accumulation of the lipids with high unsaturation degrees mostly appears in phospholipids and glycolipids for both mosses. The results also indicate that two unusual lipid classes in plants, sulfonolipids and phosphatidylmethanol are biosynthesized by the bryophytes. This has not been seen previously and show that bryophytes have a very diverse chemistry and substantially different from other plant groups.

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.

Collagenase and Tyrosinase Inhibitory Effect of Isolated Constituents from the Moss Polytrichum formosum.

Mosses from the genus Polytrichum have been shown to contain rare benzonaphthoxanthenones compounds, and many of these have been reported to have important biological activities. In this study, extracts from Polytrichum formosum were analyzed in vitro for their inhibitory properties on collagenase and tyrosinase activity, two important cosmetic target enzymes involved respectively in skin aging and pigmentation. The 70% ethanol extract showed a dose-dependent inhibitory effect against collagenase (IC50 = 4.65 mg/mL). The methanol extract showed a mild inhibitory effect of 44% against tyrosinase at 5.33 mg/mL. Both extracts were investigated to find the constituents having a specific affinity to the enzyme targets collagenase and tyrosinase. The known compounds ohioensin A (1), ohioensin C (3), and communin B (4), together with nor-ohioensin D (2), a new benzonaphthoxanthenone, were isolated from P. formosum. Their structures were determined by mass spectrometry and NMR spectroscopy. Compounds 1 (IC50 = 71.99 µM) and 2 (IC50 = 167.33 µM) showed inhibitory activity against collagenase. Compound 1 also exhibited inhibition of 30% against tyrosinase activity at 200 µM. The binding mode of the active compounds was theoretically generated by an in-silico approach against the 3D structures of collagenase and tyrosinase. These current results present the potential application from the moss P. formosum as a new natural source of collagenase and tyrosinase inhibitors.

Bio-Guided Fractionation and Molecular Networking Reveal Fatty Acids to Be Principal Anti-Parasitic Compounds in Nordic Seaweeds.

Widespread use of antimicrobial drugs has led to high levels of drug-resistance in pathogen populations and a need for novel sources of anti-bacterial and anti-parasitic compounds. Macroalgae (seaweed) are potentially a rich source of bioactive compounds, and several species have traditionally been used as vermifuges. Here, we investigated the anti-parasitic properties of four common cold-water Nordic seaweeds; Palmaria palmata (Rhodophyta), Laminaria digitata, Saccharina latissima and Ascophyllum nodosum (Ochrophyta, Phaeophyceae). Screening of organic extracts against helminths of swine (Ascaris suum) and sheep (Teladorsagia circumcincta) revealed that S. latissima and L. digitata had particularly high biological activity. A combination of molecular networking and bio-guided fractionation led to the isolation of six compounds from extracts of these two species identified in both fermented and non-fermented samples. The six isolated compounds were tentatively identified by using MS-FINDER as five fatty acids and one monoglyceride: Stearidonic acid (1), Eicosapentaenoic acid (2), Alpha-Linolenic acid (3), Docosahexaenoic acid (4), Arachidonic acid (5), and Monoacylglycerol (MG 20:5) (6). Individual compounds showed only modest activity against A. suum, but a clear synergistic effect was apparent when selected compounds were tested in combination. Collectively, our data reveal that fatty acids may have a previously unappreciated role as natural anti-parasitic compounds, which suggests that seaweed products may represent a viable option for control of intestinal helminth infections.

From Plant to Patient: Thapsigargin, a Tool for Understanding Natural Product Chemistry, Total Syntheses, Biosynthesis, Taxonomy, ATPases, Cell Death, and Drug Development.

Thapsigargin, the first representative of the hexaoxygenated guaianolides, was isolated 40 years ago in order to understand the skin-irritant principles of the resin of the umbelliferous plant Thapsia garganica. The pronounced cytotoxicity of thapsigargin is caused by highly selective inhibition of the intracellular sarco-endoplasmic Ca2+-ATPase (SERCA) situated on the membrane of the endo- or sarcoplasmic reticulum. Thapsigargin is selective to the SERCA pump and to a minor extent the secretory pathway Ca2+/Mn2+ ATPase (SPCA) pump. Thapsigargin has become a tool for investigation of the importance of SERCA in intracellular calcium homeostasis. In addition, complex formation of thapsigargin with SERCA has enabled crystallization and structure determination of calcium-free states by X-ray crystallography. These results led to descriptions of the mechanism of action and kinetic properties of SERCA and other ATPases. Inhibition of SERCA depletes Ca2+ from the sarco- and endoplasmic reticulum provoking the unfolded protein response, and thereby has enabled new studies on the mechanism of cell death. Development of protocols for selective transformation of thapsigargin disclosed the chemistry and facilitated total synthesis of the molecule. Conversion of trilobolide into thapsigargin offered an economically feasible sustainable source of thapsigargin, which enables a future drug production. Principles for prodrug development were used by conjugating a payload derived from thapsigargin with a hydrophilic peptide selectively cleaved by proteases in the tumor. Mipsagargin was developed in order to obtain a drug for treatment of cancer diseases characterized by the presence of prostate specific membrane antigen (PSMA) in the neovascular tissue of the tumors. Even though mipsagargin showed interesting clinical effects the results did not encourage funding and consequently the attempt to register the drug has been abandoned. In spite of this disappointing fact, the research performed to develop the drug has resulted in important scientific discoveries concerning the chemistry, biosynthesis and biochemistry of sesquiterpene lactones, the mechanism of action of ATPases including SERCA, mechanisms for cell death caused by the unfolded protein response, and the use of prodrugs for cancer-targeting cytotoxins. The presence of toxins in only some species belonging to Thapsia also led to a major revision of the taxonomy of the genus.

Cellulase and Macerozyme-PEG-mediated Transformation of Moss Protoplasts.

This protocol describes the generation of protoplasts from protonemal tissue of the moss Physcomitrium patens (syn. Physcomitrella patens), using Cellulase ONOZUKA R10 and Macerozyme R10, followed by polyethylene glycol (PEG) mediated transformation. The protonemal tissue grown in liquid suspension was harvested and treated with enzyme cocktails mix of 1.5% Cellulase ONOZUKA R10 and 0.5% Macerozyme R10 to generate 1,8 million protoplasts within 3 h.

Connecting moss lipid droplets to patchoulol biosynthesis.

Plant-derived terpenoids are extensively used in perfume, food, cosmetic and pharmaceutical industries, and several attempts are being made to produce terpenes in heterologous hosts. Native hosts have evolved to accumulate large quantities of terpenes in specialized cells. However, heterologous cells lack the capacity needed to produce and store high amounts of non-native terpenes, leading to reduced growth and loss of volatile terpenes by evaporation. Here, we describe how to direct the sesquiterpene patchoulol production into cytoplasmic lipid droplets (LDs) in Physcomitrium patens (syn. Physcomitrella patens), by attaching patchoulol synthase (PTS) to proteins linked to plant LD biogenesis. Three different LD-proteins: Oleosin (PpOLE1), Lipid Droplet Associated Protein (AtLDAP1) and Seipin (PpSeipin325) were tested as anchors. Ectopic expression of PTS increased the number and size of LDs, implying an unknown mechanism between heterologous terpene production and LD biogenesis. The expression of PTS physically linked to Seipin increased the LD size and the retention of patchoulol in the cell. Overall, the expression of PTS was lower in the anchored mutants than in the control, but when normalized to the expression the production of patchoulol was higher in the seipin-linked mutants.

Biosynthesis and Industrial Production of Androsteroids.

Steroids are a group of organic compounds that include sex hormones, adrenal cortical hormones, sterols, and phytosterols. In mammals, steroid biosynthesis starts from cholesterol via multiple steps to the final steroid and occurs in the gonads, adrenal glands, and placenta. This highly regulated pathway involves several cytochrome P450, as well as different dehydrogenases and reductases. Steroids in mammals have also been associated with drug production. Steroid pharmaceuticals such as testosterone and progesterone represent the second largest category of marketed medical products. There heterologous production through microbial transformation of phytosterols has gained interest in the last couple of decades. Phytosterols being the plants sterols serve as inexpensive substrates for the production of steroid derivatives. Various genes and biochemical pathways involved in phytosterol degradation have been identified in many Rhodococcus and Mycobacterium species. Apart from an early investigation in mammals, presence of steroids such as androsteroids and progesterone has also been demonstrated in plants. Their main role is linked with growth, development, and reproduction. Even though plants share some chemical features with mammals, the biosynthesis is different, with the first C22 hydroxylation as an example. This is performed by CYP11A1 in mammals and CYP90B1 in plants. Moreover, the entire plant steroid biosynthesis is not fully elucidated. Knowing this pathway could provide new processes for the industrial biotechnological production of steroid hormones in plants.

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.

Stabilization of dhurrin biosynthetic enzymes from Sorghum bicolor using a natural deep eutectic solvent.

In recent years, ionic liquids and deep eutectic solvents (DESs) have gained increasing attention due to their ability to extract and solubilize metabolites and biopolymers in quantities far beyond their solubility in oil and water. The hypothesis that naturally occurring metabolites are able to form a natural deep eutectic solvent (NADES), thereby constituting a third intracellular phase in addition to the aqueous and lipid phases, has prompted researchers to study the role of NADES in living systems. As an excellent solvent for specialized metabolites, formation of NADES in response to dehydration of plant cells could provide an appropriate environment for the functional storage of enzymes during drought. Using the enzymes catalyzing the biosynthesis of the defense compound dhurrin as an experimental model system, we demonstrate that enzymes involved in this pathway exhibit increased stability in NADES compared with aqueous buffer solutions, and that enzyme activity is restored upon rehydration. Inspired by nature, application of NADES provides a biotechnological approach for long-term storage of entire biosynthetic pathways including membrane-anchored enzymes.

Valuable Fatty Acids in Bryophytes-Production, Biosynthesis, Analysis and Applications.

Bryophytes (mosses, liverworts and hornworts) often produce high amounts of very long-chain polyunsaturated fatty acids (vl-PUFAs) including arachidonic acid (AA, 20:4 △5,8,11,14) and eicosapentaenoic acid (EPA, 20:5 △5,8,11,14,17). The presence of vl-PUFAs is common for marine organisms such as algae, but rarely found in higher plants. This could indicate that bryophytes did not lose their marine origin completely when they landed into the non-aqueous environment. Vl-PUFA, especially the omega-3 fatty acid EPA, is essential in human diet for its benefits on healthy brain development and inflammation modulation. Recent studies are committed to finding new sources of vl-PUFAs instead of fish and algae oil. In this review, we summarize the fatty acid compositions and contents in the previous studies, as well as the approaches for qualification and quantification. We also conclude different approaches to enhance AA and EPA productions including biotic and abiotic stresses.

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.

Biosynthesis of tovarol and other sesquiterpenoids in Thapsia laciniata Rouy.

The genus Thapsia produces a wide variety of sesquiterpenoids. The Mediterranean plant Thapsia laciniata Rouy is known to have a product profile that differs from several other species in the genus. Thus, the biosynthesis of sesquiterpenoids in Thapsia laciniata Rouy was investigated. Here we describe three terpene synthases, TlTPS820, TlTPS509 and TlTPS18983. TlTPS18983 is a multi-product enzyme with farnesene as the major product, while TlTPS509 produces guaiol and bulnesol along with other major and several minor unknown products. TlTPS820 is orthologous to TgTPS2 from Thapsia garganica L. and is an epikunzeaol synthase. TgCYP76AE2 from Thapsia garganica performs a triple hydroxylation of epikunzeaol at C-12 to make dihydrocostunolide. It was therefore investigated if the cytochrome P450, TlCYP76AE4 was able to use epikunzeaol as a substrate. It was found that TlCYP76AE4 hydroxylates epikunzeaol at C-8 to yield tovarol instead of dihydrocostunolide.

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

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.