Hydroxylation and glycosylation of Δ9-tetrahydrocannabinol by Catharanthus roseus cell suspension culture

Δ9-tetrahydrocannabinol is the active constituent in Cannabis sativa, with reported analgesic, anti-emetic, anti-oxidative, neuroprotective, and anti-inflammatory activities. Δ9-THC has been used to treat a number of disease states including pain, anxiety, asthma, glaucoma, and hypertension. Poor water solubility of Δ9-THC greatly reduces its clinical effectiveness. Consequently, there is a need to modify the compound to increase its polarity and pharmaceutical efficacy. The aim of this study was to test the capability of Catharanthus roseus suspension cultured cells to convert Δ9-THC into more polar derivatives. The transformed metabolites were analyzed and isolated by HPLC. Structures of some new derivatives were proposed on the basis of molecular ion peaks and fragmentation patterns obtained from LC-MS and UV spectra obtained by HPLC, respectively. Δ9-THC was rapidly absorbed by Catharanthus roseus cultured cells and upon biotransformation new glycosylated and hydroxylated derivatives were isolated by preparative HPLC. In addition, cannabinol was detected as degradation product, including its glycosylated derivative. Based on these results, it is concluded that Catharanthus cultured cells have great potential to transform Δ9-THC into more polar derivatives and can be used for the large scale production of new cannabinoids, which can be a source of new compounds with interesting pharmacological profiles

13C-Isotope-Labeling Experiments to Study Metabolism in Catharanthus roseus

Plant metabolism is a complex network. Pathways are correlated and affect each other. Secondary metabolic pathways in plant cells are regulated strictly, and upon an intra- or extra-stimuli (e.g. stress), the metabolic fluxes will change as a response on the stimuli, for example, to protect the plant against herbivores or against microbial infections. 13C-isotope-labeling experiment has been performed on cell cultures and hairy roots of Catharanthus roseus to measure fluxes through some pathways. However, due to the complexity of the total metabolic network in an intact plant, no experiments have yet been carried on C. roseus plants. In this study, [1-13C] glucose was first applied to C. roseus seedlings grown in Murashige and Skoog’s (MS) medium. In a time course, the amount and position of 13C incorporation into the metabolites were analyzed by proton nuclear magnetic resonance (1H NMR) and 1H-13C heteronuclear single quantum coherence (HSQC) NMR. The results show that the fed 13C-isotope was efficiently incorporated into and recycled in metabolism of the intact C. roseus plant. The C. roseus plants seem to be a good system for metabolic flux analysis

Metabolic changes of salicylic acid-elicited Catharanthus roseus cell suspension cultures monitored by NMR-based metabolomics

The effect of salicylic acid (SA) on the metabolic profile of Catharanthus roseus suspension cells throughout a time course (0, 6, 12, 24, 48 and 72 h after treatment) was investigated using NMR spectroscopy and multivariate data analysis. When compared to control cell lines, SA-treated cells showed a high level of sugars (glucose and sucrose) up to 48 h after treatment, followed by a dynamic change in amino acids, phenylpropanoids, and tryptamine. Additionally, one compound—2,5-dihydroxybenzoic-5-O-glucoside—was detected solely in SA-treated cells

Metabolic alterations and distribution of five-carbon precursors in jasmonic acid-elicited Catharanthus roseus cell suspension cultures

Plant science

Jasmonic Acid Effect on the Fatty Acid and Terpenoid Indole Alkaloid Accumulation in Cell Suspension Cultures of Catharanthus roseus

The stress response after jasmonic acid (JA) treatment was studied in cell suspension cultures of Catharanthus roseus. The effect of JA on the primary and secondary metabolism was based on changes in profiles of fatty acids (FA) and terpenoid indole alkaloids (TIA). According to multivariate data analyses (MVDA), three major time events were observed and characterized according to the variations of specific FA and TIA: after 0–30 min of induction FA such as C18:1, C20:0, C22:0 and C24:0 were highly induced by JA; 90–360 min after treatment was characterized by variations of C14:0 and C15:0; and 1440 min after induction JA had the largest effect on both group of metabolites were C18:1, C18:2, C18:3, C16:0, C20:0, C22:0, C24:0, catharanthine, tabersonine-like 1, serpentine, tabersonine and ajmalicine-like had the most significant variations. These results unambiguously demonstrate the profound effect of JA particularly on the accumulation of its own precursor, C18:3 and the accumulation of TIA, which can be considered as late stress response events to JA since they occurred only after 1440 min. These observations show that the early events in the JA response do not involve the de novo biosynthesis of neither its own precursor nor TIA, but is due to an already present biochemical system

Metabolic alteration of Catharanthus roseus cell suspension cultures overexpressing geraniol synthase in the plastids or cytosol

Previous studies showed that geraniol could be an upstream limiting factor in the monoterpenoid pathway towards the production of terpenoid indole alkaloid (TIA) in Catharanthus roseus cells and hairy root cultures. This shortage in precursor availability could be due to (1) limited expression of the plastidial geraniol synthase resulted in a low activity of the enzyme to catalyze the conversion of geranyl diphosphate to geraniol; or (2) the limitation of geraniol transport from plastids to cytosol. Therefore, in this study, C. roseus’s geraniol synthase (CrGES) gene was overexpressed in either plastids or cytosol of a non-TIA producing C. roseus cell line. The expression of CrGES in the plastids or cytosol was confirmed and the constitutive transformation lines were successfully established. A targeted metabolite analysis using HPLC shows that the transformed cell lines did not produce TIA or iridoid precursors unless elicited with jasmonic acid, as their parent cell line. This indicates a requirement for expression of additional, inducible pathway genes to reach production of TIA in this cell line. Interestingly, further analysis using NMR-based metabolomics reveals that the overexpression of CrGES impacts primary metabolism differently if expressed in the plastids or cytosol. The levels of valine, leucine, and some metabolites derived from the shikimate pathway, i.e. phenylalanine and tyrosine were significantly higher in the plastidial- but lower in the cytosolic-CrGES overexpressing cell lines. This result shows that overexpression of CrGES in the plastids or cytosol caused alteration of primary metabolism that associated to the plant cell growth and development. A comprehensive omics analysis is necessary to reveal the full effect of metabolic engineering

Monoterpenoid indole alkaloids biosynthesis and its regulation in Catharanthus roseus: a literature review from genes to metabolites

Plant science