Biosynthetic pathway of prescription cucurbitacin IIa and high-level production of key triterpenoid intermediates in engineered yeast and tobacco.

Cucurbitacin IIa is a triterpenoid isolated exclusively from Hemsleya plants and a non-steroidal anti-inflammatory drug that functions as the main ingredient of prescription Hemslecin capsules and tablets in China. Synthetic biology provides new strategies for production of such valuable cucurbitacins at a large scale; however, the biosynthetic pathway of cucurbitacin IIa has been unknown, and the heterologous production of cucurbitacins in galactose medium has been expensive and low yielding. In this study, we characterized the functions of genes encoding two squalene epoxidases (HcSE1-2), six oxidosqualene cyclases (HcOSC1-6), two CYP450s (HcCYP87D20 and HcCYP81Q59), and an acyltransferase (HcAT1) in cucurbitacin IIa biosynthesis by heterologous expression in Saccharomyces cerevisiae and Nicotiana benthamiana. We achieved high-level production of the key cucurbitacin precursor 11-carbonyl-20ß-hydroxy-Cuol from glucose in yeast via modular engineering of the mevalonate pathway and optimization of P450 expression levels. The resulting yields of 46.41 mg/l 11-carbonyl-20ß-hydroxy-Cuol and 126.47 mg/l total cucurbitacin triterpenoids in shake flasks are the highest yields yet reported from engineered microbes. Subsequently, production of 11-carbonyl-20ß-hydroxy-Cuol by transient gene expression in tobacco resulted in yields of 1.28 mg/g dry weight in leaves. This work reveals the key genes involved in biosynthesis of prescription cucurbitacin IIa and demonstrates that engineered yeast cultivated with glucose can produce high yields of key triterpenoid intermediates. We describe a low-cost and highly efficient platform for rapid screening of candidate genes and high-yield production of pharmacological triterpenoids.

Lessons learned from metabolic engineering in hairy roots: Transcriptome and metabolic profile changes caused by Rhizobium-mediated plant transformation in Cucurbitaceae species.

Cucurbitaceae species are used in traditional medicine around the world. Cucurbitacins are highly oxygenated triterpenoids found in Cucurbitaceae species and exhibit potent anticancer activity alone and in combination with other existing chemotherapeutic drugs. Therefore, increasing production of these specialized metabolites is of great relevance. We recently showed that hairy roots of Cucurbita pepo can be used as a platform for metabolic engineering of cucurbitacins to modify their structure and increase their production. To study the changes in cucurbitacin accumulation upon formation of hairy roots, an empty vector (EV) control and Cucurbitacin inducing bHLH transcription factor 1 (CpCUCbH1)-overexpressing hairy roots of C. pepo were compared to untransformed (WT) roots. Whilst CpCUCbH1-overexpression increased production of cucurbitacins I and B by 5-fold, and cucurbitacin E by 3-fold when compared to EV lines, this increase was not significantly different when compared to WT roots. This indicated that Rhizobium rhizogenes transformation lowered the cucurbitacins levels in hairy roots, but that increasing expression of cucurbitacin biosynthetic genes by CpCUCbH1-overexpression restored cucurbitacin production to WT levels. Subsequent metabolomic and RNA-seq analysis indicated that the metabolic profile and transcriptome of hairy roots was significantly changed when compared to WT roots. Interestingly, it was observed that 11% of the differentially expressed genes were transcription factors. It was noteworthy that the majority of transcripts showing highest Pearson correlation coefficients to the Rhizobium rhizogenes genes rolB, rolC and ORF13a, were predicted to be transcription factors. In summary, hairy roots are an excellent platform for metabolic engineering of plant specialized metabolites, but these extensive transcriptome and metabolic profile changes should be considered in subsequent studies.

Vacuolar MATE/DTX protein-mediated cucurbitacin C transport is co-regulated with bitterness biosynthesis in cucumber.

Membrane-localized transporters constitute important components for specialized metabolism in plants. However, due to the vast array of specialized metabolites produced by plants, and the large families of transporter genes, knowledge about the intracellular and intercellular transport of plant metabolites is still in its infancy. Cucurbitacins are bitter and defensive triterpenoids produced mainly in the cucurbits. Using a comparative genomics and multi-omics approach, a MATE gene (CsMATE1), physically clustered with cucurbitacin C (CuC) biosynthetic genes, was identified and functionally shown to sequester CuC in cucumber leaf mesophyll cells. Notably, the CuC transport process is strictly co-regulated with CuC biosynthesis. CsMATE1 clustering with bitterness biosynthesis genes may provide benefits and a basis for this feedback regulation on CuC sequestration and biosynthesis. Identification of transport systems for plant-specialized metabolites can accelerate the metabolic engineering of high-value-added compounds by simplifying their purification process.

Comparative transcriptome analysis and identification of candidate genes involved in cucurbitacin IIa biosynthesis in Hemsleya macrosperma.

Hemsleya macrosperma (H. macrosperma) is widely used in southwestern China as folk medicine with various bioactivities. Cucurbitacin IIa is the main active component in H. macrosperma and draws increased attention for its potential pharmacological activities. In order to reveal the mechanism of cucurbitacin IIa biosynthesis and regulation in H. macrosperma, transcriptome analysis was performed to compare differentially expressed genes in three tissues (root tuber, stem and leaf). A total of 47 946 unigenes were generated from these tissues and 55 unigenes were identified as candidate genes involved in triterpenoid backbone biosynthesis. Three homologous genes encoding squalene epoxidase (HmSE) were discovered and successfully expressed in a prokaryotic system. HmSE1 was found to be responsible for oxidization of squalene. In addition, several cytochrome P450s and transcription factors were predicted as candidates associated to cucurbitacin IIa biosynthesis. Notably, the expression profiles of those putative genes showed a positive correlation with elevated curcurbitacin IIa production in methyl jasmonate-elicited suspension cells of H. macrosperma., suggesting probable functions of the candidates on curcurbitacin IIa biosynthesis. These findings provide insights on cucurbitacin IIa biosynthesis and regulation in H. macrosperma.

Cucurbitacins Elicit Anti-Platelet Activity via Perturbation of the Cytoskeleton and Integrin Function.

Cucurbitacins are dietary compounds that have been shown to elicit a range of anti-tumour, anti-inflammatory and anti-atherosclerotic activities. Originally identified as signal transducer and activator of transcription, STAT, inhibitors, a variety of mechanisms of action have since been described, including dysregulation of the actin cytoskeleton and disruption of integrin function. Integrin outside-in signalling and cytoskeletal rearrangements are critical for the propagation of stable thrombus formation and clot retraction following platelet adhesion at the site of vessel damage. The effects of cucurbitacins on platelet function and thrombus formation are unknown. We report for the first time anti-platelet and anti-thrombotic effects of cucurbitacins B, E and I in human platelets. Treatment of platelets with cucurbitacins resulted in attenuation of platelet aggregation, secretion and fibrinogen binding following stimulation by platelet agonists. Cucurbitacins were also found to potently inhibit other integrin- and cytoskeleton-mediated events, including adhesion, spreading and clot retraction. Further investigation of cytoskeletal dynamics found treatment with cucurbitacins altered cofilin phosphorylation, enhanced activation and increased F actin polymerisation and microtubule assembly. Disruption to cytoskeletal dynamics has been previously shown to impair integrin activation, platelet spreading and clot retraction. Anti-platelet properties of cucurbitacins were found to extend to a disruption of stable thrombus formation, with an increase in thrombi instability and de-aggregation under flow. Our research identifies novel, anti-platelet and anti-thrombotic actions of cucurbitacins that appear to be linked to dysregulation of cytoskeletal dynamics and integrin function.

Identification of seven undescribed cucurbitacins in Cucumis sativus (cucumber) and their cytotoxic activity.

Cucurbitacin C-type cucurbitacins that are only identified in Cucumis sativus (cucumber) are, in part, responsible for the health benefits and bitter flavor. Nevertheless, detailed information about those functional ingredients in cucumber is scarce. In this study, ten cucurbitacin C analogues including seven undescribed ones have been isolated from the bitter leaves of cucumber, in which six compounds showed growth inhibition capabilities against tumor cell lines HepG2, A549, DU145 and HCT116. Intriguingly, cucurbitacin C6 and C7 exhibited a significant inhibition effect compared to the positive control taxol (IC50 = 1.86 ± 0.17 µM) on HepG2 cell line with IC50 values of 10.06 ± 0.34 µM and 4.16 ± 0.42 µM, respectively. The mechanism of cucurbitacin-induced apoptosis is likely down-regulating the expression of caspase-related proteins. This work enlarges the knowledge of the cucurbitacins in cucumber and highlights the importance of cucumber as a source of specialized metabolites in the food and medicinal industries.

Morphometric and total protein responses in Meloidogyne incognita second-stage juveniles to Nemafric-BL phytonematicide.

After hatch, second-stage juveniles (J2) of root-knot (Meloidogyne species) nematodes could spend at least 12 weeks in soil solutions searching for penetration sites of suitable host plants. The external covering of nematodes, the cuticle, consists of various layers that contain glycoproteins, lipids, soluble proteins (collagens) and insoluble proteins (cuticulins). Generally, cucurbitacins are lipophilic, but there is scant information on how cuticular proteins relate to these complex terpenoids. A study was conducted to investigate the nature and extent of damage post-exposure of J2 to a wide range of Nemafric-BL phytonematicide concentrations. Post-72 h exposure to Nemafric-BL phytonematicide, nematode morphometrics versus phytonematicides exhibited either negative quadratic, positive quadratic, or negative linear relations, with the models explained by significant (P < 0.05) associations (R-squared). Similarly, total proteins versus phytonematicide exhibited significant negative quadratic relations. The principal component analysis indicated that concentration level of 2-4% of Nemafric-BL phytonematicide have the highest impact on the morphometric changes of J2. In conclusion, the nature and extent of damage suggested that Nemafric-BL phytonematicide was highly nematicidal as opposed to being nematostatic, thereby explaining its potent suppressive effects on nematode population densities.

Divergence of defensive cucurbitacins in independent Cucurbita pepo domestication events leads to differences in specialist herbivore preference.

Crop domestication and improvement often concurrently affect plant resistance to pests and production of secondary metabolites, creating challenges for isolating the ecological implications of selection for specific metabolites. Cucurbitacins are bitter triterpenoids with extreme phenotypic differences between Cucurbitaceae lineages, yet we lack integrated models of herbivore preference, cucurbitacin accumulation, and underlying genetic mechanisms. In Cucurbita pepo, we dissected the effect of cotyledon cucurbitacins on preference of a specialist insect pest (Acalymma vittatum) for multiple tissues, assessed genetic loci underlying cucurbitacin accumulation in diverse germplasm and a biparental F2 population (from a cross between two independent domesticates), and characterized quantitative associations between gene expression and metabolites during seedling development. Acalymma vittatum affinity for cotyledons is mediated by cucurbitacins, but other traits contribute to whole-plant resistance. Cotyledon cucurbitacin accumulation was associated with population structure, and our genetic mapping identified a single locus, Bi-4, containing genes relevant to transport and regulation - not biosynthesis - that diverged between lineages. These candidate genes were expressed during seedling development, most prominently a putative secondary metabolite transporter. Taken together, these findings support the testable hypothesis that breeding for plant resistance to insects involves targeting genes for regulation and transport of defensive metabolites, in addition to core biosynthesis genes.

Metabolite Profiling and Quantitation of Cucurbitacins in Cucurbitaceae Plants by Liquid Chromatography coupled to Tandem Mass Spectrometry.

Cucurbitaceae is an important plant family because many of its species are consumed as food, and used in herbal medicines, cosmetics, etc. It comprises annual vines and is rich in various bioactive principles which include the cucurbitacins. These steroidal natural products, derived from the triterpene cucurbitane, are mainly the bitter principles of the family Cucurbitaceae. Their biological activities include anti-inflammatory, hepatoprotective, and anti-cancer activities. A total of 10 species belonging to 6 genera of the Cucurbitaceae family along with Cissampelos pareira (Menispermaceae) were included in this study. A comprehensive profiling of certain natural products was developed using HPLC-QTOF-MS/MS analysis and a distribution profile of several major natural products in this family was obtained. A total of 51 natural products were detected in both positive and negative ionization modes, based on accurate masses and fragmentation patterns. Along with this, quantitation of four bioactive cucurbitacins, found in various important plants of the Cucurbitaceae family, was carried out using multiple reaction monitoring (MRM) approach on an ion trap mass spectrometer. Cucurbitacin Q was found to be the most abundant in C. pareira, while Citrullus colocynthis contained all four cucurbitacins in abundant quantities. The developed quantitation method is simple, rapid, and reproducible.

Biological screening of cucurbitacin inspired estrone analogs targeting mitogen-activated protein kinase (MAPK) pathway.

Assembly of cucurbitacin inspired estrone analogs has been previously synthesized and screened against melanoma cell lines. Further synthetic optimization was executed via installation of Azide polar functional moiety across 23, 24 α, ß-unsaturated ketone side chain using Michael addition reaction. This was followed by biological screening against melanoma cell lines employing MTT assay, in-cell-based ELISA assay, and Western blot analysis to monitor the potential of the synthesized analogs to inhibit the phosphorylated ERK levels. This resulted in evolution of MH-4 possessing IC50 of 3.59 µm with significant decrease in the p-ERK and targeting MAPK pathway.

Surface plasmon resonance and circular dichroism characterization of cucurbitacins binding to serum albumins for early pharmacokinetic profiling.

Cucurbitacins are a group of tetracyclic triterpenoids, known for centuries for their anti-cancer and anti-inflammatory properties, which are being actively investigated over the past decades in order to elucidate their mechanism of action. In perspective of being used as therapeutic molecules, a pharmacokinetic characterization is crucial to assess the affinity toward blood carrier proteins and extrapolate distribution volumes. Usually, pharmacokinetic data are first collected on animal models and later translated to humans; therefore, an early characterization of the interaction with carrier proteins from different species is highly desirable. In the present study, the interactions of cucurbitacins E and I with human and rat serum albumins (HSA and RSA) were investigated by means of surface plasmon resonance (SPR)-based optical biosensing and circular dichroism (CD) spectroscopy. Active HSA and RSA sensor chip surfaces were prepared through an amine coupling reaction protocol, and the equilibrium dissociation constants (Kd) for the different cucurbitacins-serum albumins complexes were then determined by SPR analysis. Further information on the binding of cucurbitacins to serum albumins was obtained by CD competition experiments with biliverdin, a specific marker binding to subdomain IB of HSA. SPR data unveiled a previously unreported binding event between CucI and HSA; the determined binding affinities of both compounds were slightly higher for RSA with respect to HSA, even though all the compounds can be ranked as high-affinity binders for both carriers. CD analysis showed that the two cucurbitacins modify the binding of biliverdin to serum albumins through opposite allosteric modulation (positive for HSA, negative for RSA), confirming the need for caution in the translation of pharmacokinetic data across species.

The effect of red light and far-red light conditions on secondary metabolism in agarwood.

BACKGROUND: Agarwood, a heartwood derived from Aquilaria trees, is a valuable commodity that has seen prevalent use among many cultures. In particular, it is widely used in herbal medicine and many compounds in agarwood are known to exhibit medicinal properties. Although there exists much research into medicinal herbs and extraction of high value compounds, few have focused on increasing the quantity of target compounds through stimulation of its related pathways in this species. RESULTS: In this study, we observed that cucurbitacin yield can be increased through the use of different light conditions to stimulate related pathways and conducted three types of high-throughput sequencing experiments in order to study the effect of light conditions on secondary metabolism in agarwood. We constructed genome-wide profiles of RNA expression, small RNA, and DNA methylation under red light and far-red light conditions. With these profiles, we identified a set of small RNA which potentially regulates gene expression via the RNA-directed DNA methylation pathway. CONCLUSIONS: We demonstrate that light conditions can be used to stimulate pathways related to secondary metabolism, increasing the yield of cucurbitacins. The genome-wide expression and methylation profiles from our study provide insight into the effect of light on gene expression for secondary metabolism in agarwood and provide compelling new candidates towards the study of functional secondary metabolic components.

Attracting mutualists and antagonists: plant trait variation explains the distribution of specialist floral herbivores and pollinators on crops and wild gourds.

UNLABELLED: • PREMISE OF THE STUDY: Floral traits play important roles in pollinator attraction and defense against floral herbivory. However, plants may experience trade-offs between conspicuousness to pollinators and herbivore attraction. Comparative studies provide an excellent framework to examine the role of multiple traits shaping mutualist and antagonist interactions.• METHODS: To assess whether putative defensive and attractive traits predict species interactions, we grew 20 different Cucurbitaceae species and varieties in the field to measure interactions with pollinators and herbivores and in the greenhouse to assess trait variation. Cucurbits are characterized by the production of cucurbitacins, bitter nonvolatile terpenoids that are effective against generalist herbivores but can attract specialist beetles. We determined whether plant traits such as cucurbitacins predict herbivore resistance and pollinator attraction using an information-theoretic approach.• KEY RESULTS: Mutualists and floral antagonists were attracted to the same cucurbit varieties once they flowered. However, rather than cucurbitacin concentration, we found that the size of the flower and volatile emissions of floral sesquiterpenoids explained both pollinator and floral herbivore visitation preference across cucurbit taxa. This pattern held across cucurbit taxa and within the Cucurbita genus.• CONCLUSIONS: Surprisingly, floral sesquiterpenoid volatiles, which are associated with direct defense, indirect defense, and attraction, rather than defense traits such as cucurbitacins, appeared to drive interactions with both pollinators and floral herbivores across cucurbit taxa. Identifying the relevant plant traits for attraction and deterrence is important in this economically valuable crop, particularly if pollinators and floral herbivores use the same plant traits as cues.

Identification of cucurbitacins and assembly of a draft genome for Aquilaria agallocha.

BACKGROUND: Agarwood is derived from Aquilaria trees, the trade of which has come under strict control with a listing in Appendix II of the Convention on International Trade in Endangered Species of Wild Fauna and Flora. Many secondary metabolites of agarwood are known to have medicinal value to humans, including compounds that have been shown to elicit sedative effects and exhibit anti-cancer properties. However, little is known about the genome, transcriptome, and the biosynthetic pathways responsible for producing such secondary metabolites in agarwood. RESULTS: In this study, we present a draft genome and a putative pathway for cucurbitacin E and I, compounds with known medicinal value, from in vitro Aquilaria agallocha agarwood. DNA and RNA data are utilized to annotate many genes and protein functions in the draft genome. The expression changes for cucurbitacin E and I are shown to be consistent with known responses of A. agallocha to biotic stress and a set of homologous genes in Arabidopsis thaliana related to cucurbitacin bio-synthesis is presented and validated through qRT-PCR. CONCLUSIONS: This study is the first attempt to identify cucurbitacin E and I from in vitro agarwood and the first draft genome for any species of Aquilaria. The results of this study will aid in future investigations of secondary metabolite pathways in Aquilaria and other non-model medicinal plants.

Structural optimization and biological screening of a steroidal scaffold possessing cucurbitacin-like functionalities as B-Raf inhibitors.

Inhibition of the mitogen-activated protein kinase (MAPK) pathway by targeting the commonly occurring mutated B-Raf in melanoma has become a practical method for the development of drugs and drug candidates. In order to expand upon the currently reported structural scaffolds used to target the MAPK pathway, molecular docking studies led to the installation an α,ß-unsaturated ketone side chain, related to the cucurbitacin class of natural products, on to an estrone core via an aldol condensation reaction, along with installation of the Δ(9,11) olefin to assemble what has been defined as a pseudo-cis configuration at the B/C ring juncture. Combination of these cucurbitacin-like features resulted in a compound with an enhanced biological profile against the A-375 mutant B-Raf cell line, in regards to their cytotoxicity and inhibitory activity toward phosphorylated extracellular-signal-regulated kinase (ERK).