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Sesquiterpene lactones - such as those found in chicory - are considered promising bioactive compounds. These small molecules have shown several health benefits for various diseases, including brain disorders. However, it is unknown whether these compounds can cross the blood-brain barrier (BBB), and which could be the effects on brain microvascular endothelial cells. We show that six sesquiterpene lactones evaluated in an in vitro model of the BBB have different capacities to be transported through the barrier. Costunolide presented more than 20 % of transport while lactucin, 11β-13-dihydrolactucin, 11β-13-dihydrolactucopicrin, and parthenolide presented between 10 % and 20 %, whilst almost no transport was detected for lactucopicrin. Furthermore, costunolide and parthenolide reduced P-gp ABC transporter expression alongside an increase in caveolin-1, the main protein of caveolae. Remarkably, these two compounds improved barrier tightness by increasing the expression of both tight and adherens junctions. These findings open a new avenue to explore costunolide and parthenolide as promising compounds for brain therapies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

SARS-CoV-2 is a highly transmissible and pathogenic coronavirus causing a respiratory disease that emerged in 2019, leading to a public health emergency situation which continues to date. The treatment options are still very limited and vaccines available are less effective against new variants. SARS-CoV-2 enzymes, namely main protease (Mpro) and papain-like protease (PLpro), play a pivotal role in the viral life cycle, making them a putative drug target. Here, we described for the first time the potential inhibitory activity of chicory extract against both proteases. Besides, we have identified that the four most abundant sesquiterpene lactones in chicory inhibited these proteases, showing an effective binding in the active sites of Mpro and PLpro. This paper provides new insight for further drug development or food-based strategies for the prevention of SARS-CoV-2 by targeting viral proteases.

Chicory (Cichorium intybus var. sativum) is an industrial crop species cultivated for the production of a fructose polymer inulin, which is used as a low-calorie sweetener and prebiotic. Besides, inulin chicory taproots also accumulate sesquiterpene lactones (STLs). These are bitter tasting compounds, which need to be removed during inulin extraction, resulting in additional costs. In this work, we describe chicory lines where STL accumulation is almost completely eliminated. Genome editing using the CRISPR/Cas9 system was used to inactivate four genes that encode the enzyme that performs the first dedicated step in STL synthesis, germacrene A synthase (CiGAS). Chicory lines were obtained that carried null mutations in all four CiGAS genes. Lines lacking functional CiGAS alleles showed a normal phenotype upon greenhouse cultivation and show nearly complete elimination of the STL synthesis in the roots. It was shown that the reduction in STLs could be attributed to mutations in genetically linked copies of the CiGAS-short gene and not the CiGAS-long gene, which is relevant for breeding the trait into other cultivars. The inactivation of the STL biosynthesis pathway led to increase in phenolic compounds as well as accumulation of squalene in the chicory taproot, presumably due to increased availability of farnesyl pyrophosphate (FFP). These results demonstrate that STLs are not essential for chicory growth and that the inhibition of the STL biosynthesis pathway reduced the STL levels chicory which will facilitate inulin extraction., (© 2021 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

Cichorium intybus L . or chicory plants are a natural source of health-promoting compounds in the form of supplements such as inulin, as well as other bioactive compounds such as sesquiterpene lactones (SLs). After inulin extraction, chicory roots are considered waste, with most SLs not being harnessed. We developed and optimized a new strategy for SL extraction that can contribute to the conversion of chicory root waste into valuable products to be used in human health-promoting applications. In our work, rich fractions of SLs were recovered from chicory roots using supercritical CO 2 . A response surface methodology was used to optimize the process parameters (pressure, temperature, flow rate, and co-solvent percentage) for the extraction performance. The best operating conditions were achieved at 350 bar, 40 °C, and 10% EtOH as a co-solvent in a 15 g/min flow rate for 120 min. The extraction with supercritical CO 2 revealed to be more selective for the SLs than the conventional solid-liquid extraction with ethyl acetate. In our work, 1.68% mass and a 0.09% sesquiterpenes yield extraction were obtained, including the recovery of two sesquiterpene lactones (8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin), which, to the best of our knowledge, are not commercially available. A mixture of the abovementioned compounds were tested at different concentrations for their toxic profile and anti-inflammatory potential towards a human calcineurin/NFAT orthologue pathway in a yeast model, the calcineurin/Crz1 pathway. The SFE extract obtained, rich in SLs, yielded results of inhibition of 61.74 ± 6.87% with 50 µg/mL, and the purified fraction containing 8-deoxylactucin and 11β,13-dihydro-8-deoxylactucin inhibited the activation of the reporter gene up to 53.38 ± 3.9% at 10 µg/mL. The potential activity of the purified fraction was also validated by the ability to inhibit Crz1 nuclear translocation and accumulation. These results reveal a possible exploitable green technology to recover potential anti-inflammatory compounds from chicory roots waste after inulin extraction.

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, β-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Plant sesquiterpenes, such as (+)-valencene, artemisinin, and farnesene are valuable chemicals for use as aromatics, pharmaceuticals, and biofuels. Plant-based production systems for terpenoids critically depend on the availability of farnesyl diphosphate (FPP). Currently, these systems show insufficient yields, due to the competition for FPP of newly introduced pathways with endogenous ones. In this study, for the first time an RNAi strategy aiming at silencing of endogenous pathways for increased (+)-valencene production was employed. Firstly, a transient production system for (+)-valencene in Nicotiana benthamiana was set up using agroinfiltration. Secondly, silencing of the endogenous 5-epi-aristolochene synthase (EAS) and squalene synthase (SQS) that compete for the FPP pool was deployed. This resulted in a N. benthamiana plant that produces (+)-valencene as a prevalent volatile with a 2.8-fold increased yield. Finally, the size of the FPP pool was increased by overexpression of enzymes that are rate-limiting in FPP biosynthesis. Combined with silencing of EAS and SQS, no further increase of (+)-valencene production was observed, but emission of farnesol. Formation of farnesol, which is a breakdown product of FPP, indicates that overproducing sesquiterpenes is no longer limited by FPP availability in the cytosol. This study shows that metabolic engineering of plants can effectively be used for increased production of desired products in plants., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Production of commercially interesting sesquiterpenes was previously examined in plants and microorganisms such as Escherichia coli and Saccharomyces cerevisiae. We here investigate the potential of the mushroom Schizophyllum commune for the production of sesquiterpenes. Genomic analysis of S. commune revealed that the mevalonate pathway required for the synthesis of the farnesyl diphosphate substrate for sesquiterpene production is operational. Introduction of a valencene synthase gene resulted in production of the sesquiterpene (+)-valencene, both in mycelium and in fruiting bodies. Levels of (+)-valencene in culture media of strains containing a mutated RGS regulatory protein gene (thn) were increased fourfold compared to those in wild-type transformants. Up to 16 mg L(-1) (+)-valencene was produced in these strains. In addition, the amount of (+)-valencene containing n-dodecane recovered from the culture medium increased sixfold to sevenfold in the thn mutant strains due to the absence of schizophyllan.

Nootkatone is one of the major terpenes in the heartwood of the Nootka cypress Callitropsis nootkatensis. It is an oxidized sesquiterpene, which has been postulated to be derived from valencene. Both valencene and nootkatone are used for flavouring citrus beverages and are considered among the most valuable terpenes used at commercial scale. Functional evaluation of putative terpene synthase genes sourced by large-scale EST sequencing from Nootka cypress wood revealed a valencene synthase gene (CnVS). CnVS expression in different tissues from the tree correlates well with nootkatone content, suggesting that CnVS represents the first dedicated gene in the nootkatone biosynthetic pathway in C. nootkatensis The gene belongs to the gymnosperm-specific TPS-d subfamily of terpenes synthases and its protein sequence has low similarity to known citrus valencene synthases. In vitro, CnVS displays high robustness under different pH and temperature regimes, potentially beneficial properties for application in different host and physiological conditions. Biotechnological production of sesquiterpenes has been shown to be feasible, but productivity of microbial strains expressing valencene synthase from Citrus is low, indicating that optimization of valencene synthase activity is needed. Indeed, expression of CnVS in Saccharomyces cerevisiae indicated potential for higher yields. In an optimized Rhodobacter sphaeroides strain, expression of CnVS increased valencene yields 14-fold to 352 mg/L, bringing production to levels with industrial potential., (© 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Chicory (Cichorium intybus L.), which is known to have a variety of terpene-hydroxylating activities, was screened for a P450 mono-oxygenase to convert (+)-valencene to (+)-nootkatone. A novel P450 cDNA was identified in a chicory root EST library. Co-expression of the enzyme with a valencene synthase in yeast, led to formation of trans-nootkatol, cis-nootkatol and (+)-nootkatone. The novel enzyme was also found to catalyse a three step conversion of germacrene A to germacra-1(10),4,11(13)-trien-12-oic acid, indicating its involvement in chicory sesquiterpene lactone biosynthesis. Likewise, amorpha-4,11-diene was converted to artemisinic acid. Surprisingly, the chicory P450 has a different regio-specificity on (+)-valencene compared to germacrene A and amorpha-4,11-diene., (Copyright © 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

Highlights: [•] (+)-Nootkatone is a major sesquiterpene component of Alaska cedar essential oil. [•] Alaska cedar valencene oxidase CYP706M1 was identified and cloned. [•] CYP706M1 catalyses the oxidation of (+)-valencene in yeast and in vitro. [•] (+)-Nootkatone was produced at 144μg/L by CYP706M1-expressing yeast culture. [•] CYP706M1 is the first valencene oxidase from a (+)-nootkatone producing plant. [Copyright &y& Elsevier]

Background: Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings: Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3- methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg.kg-1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance: This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed. [ABSTRACT FROM AUTHOR]

Highlights • In this work we study the genomics of biosynthesis of bioactive sesquiterpene lactones in Chicory. • Attention was focused on germacrene A synthases, mediating the first dedicated step in sesquiterpene lactone biosynthesis. • Genomic regions covering gene sequences for six germacrene A synthases were identified in a BAC library. • Importance of identified genomic regions was addressed in leaf and taproot parts, by promoter-GFP analysis in chicory. Abstract Chicory (Cichorium intybus L.) produces bitter sesquiterpene lactones (STLs). Some enzymes in the biosynthetic pathway towards these compounds have been characterized. However, the genomic organization and tissue specificity of their biosynthesis is largely unknown. Concentrations of two sesquiterpene lactones and expression of genes involved in the first dedicated biosynthetic step were measured in different chicory tissues. BAC clones containing different genes encoding germacrene A synthase were sequenced, and revealed several tightly linked paralogs. Promoters of genes encoding two germacrene A synthases were fused to GFP and expressed in plants regenerated from transformed chicory hairy root cultures. Highest expression was observed in the epidermis of leaves and external root tissue. This work opens the possibility to select for chicory germplasm diversified in STL content, and to study their role in chicory in defence and physiology. [ABSTRACT FROM AUTHOR]