THCA Synthase Research Articles

Genetic Characterization and Chemical Identification of Moroccan Cannabis sativa (L.) Seeds: Extraction, and In Vitro and In Silico Biological Evaluation.

This study investigated the molecular, phytochemical, and biological aspects of ten local Moroccan traditional landrace Cannabis seeds. Genetic polymorphisms were analyzed using DNA barcode determination, revealing two distinct molecular profiles: "Cannabis, species sativa, subspecies indica" and "Cannabis, species sativa, subspecies sativa". Furthermore, a new sequence was identified by sequencing of the THCA synthase coding gene. Chemical profiling via HPLC-ESI-FULL-MS and GC-MS-MS of AMSD1 maceration extracts revealed 13 non-volatile chemicals, including 3 inactive cannabinoids and 3 polyphenols, and 24 intriguing volatile compounds, including 7 previously unreported in Cannabis seed extracts. Moreover, the in vitro/in silico analysis provision of biological activities through their antioxidant power, antimicrobial effect, and cytotoxicity potency, as well as antiviral activity, were realized. These results contribute to a thorough comprehension of Moroccan Cannabis seeds, illuminating their molecular, phytochemical, and biological features. Furthermore, they highlight the seeds as a potential source of nutritious components with antioxidant properties, offering valuable insights for future research.

In silico and in vitro analysis of THCA synthase gene in Moroccan Cannabis sativa, L

Background: Cannabis ( Cannabis sativa, L.) is an archaic, most infamous plant with unique therapeutic characteristics, and great economic interest. The identification of its THCAS synthesizing enzyme was a great leap forward in Cannabis investigation. However, basic molecular biology for THCAS gene expression remains largely unstudied. In this paper, we explored the ability of bioinformatics resources in the design of molecular cloning protocol and prediction of three-dimensional structure of recombinant THCAS in Moroccan Cannabis sativa variety. Methods: We described conventional experiments performed in vitro. We used specific primers to isolate the nucleotide sequence of THCAS from the leaves of Cannabis, without including the native signal sequence.we prepared the gene by amors containing specific adapters to integrate the gene into the expression vector PpinkαHC, transforming E.coli bacteria, in order to express the gene in Pichia pasteri yeast. Results: As a result, we found high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported. In addition, we predicted the structure of the recombinant THCAS using the organic crystal structure prediction method, the amino acid sequence reference, and the artificial intelligence technology. Conclusions: In this study, we have successfuly cloned recombinant THCAS enzyme in E.Coli for further expression in host Pichia pastori.

In silico and in vitro analysis of THCA synthase gene in Moroccan Cannabis Sativa, L

Background: Cannabis (Cannabis sativa, L.) is an archaic, most infamous plant with unique therapeutic characteristics, and great economic interest. The identification of its THCAS synthesizing enzyme was a great leap forward in Cannabis investigation. However, basic molecular biology for THCAS gene expression remains largely unstudied. Methods: In this paper, we explored the ability of bioinformatics resources in the design of molecular cloning protocol and prediction of three-dimensional structure of recombinant THCAS in Moroccan Cannabis sativa variety. We further described conventional experiments performed in vitro. We used specific markers to isolate the nucleotide sequence of THCAS from the leaves of Cannabis, without including the native signal sequence. Results: As a result, we found high sequence similarity with THCAS and the mRNA precursor of the same gene as previously reported. In addition, we predicted the structure of the recombinant THCAS using the organic crystal structure prediction method, the amino acid sequence reference, and the artificial intelligence technology. Conclusions: This study paves the way to successfully express recombinant THCAS enzyme in hosts like Pichia pastori.

Delta-9-Tetrahydrocannabinolic Acid B: A Mechanism for its Formation in Cannabis.

There appears to be consensus among Cannabis biologists that delta-9-tetrahydrocannabinolic acid A (THCA-A) is the exclusive product of the enzyme THCA synthase. This then leaves an open question for formation of the THCA-A structural isomer, delta-9-tetrahydrocannabinolic acid B (THCA-B), discovered as a minor product in Cannabis in 1969. With no reasonable biochemical pathway to explain the presence of THCA-B in Cannabis, a synthetic route was next considered. Using established literature precedent, a photochemical mechanism has been proposed for the conversion of THCA-A to cannabidiolic acid (CBDA), followed by conversion of CBDA to THCA-B employing bond-breaking and bond-forming reactions.

Overview of Cannabis including Kampo Medicine and Therapy for Treatment of Dementia: A Review.

Cannabis sativa L. is an annual herb oldest cultivated plants as a source of fiber since about 5000 B.C. On the other hand, the cannabis flower and seed are listed in Shennong’s classic Materia Medica approximately 2000 years ago. The formulas prescribed with cannabis in Kampo medicine have been summarized. Cannabidiol (CBD) and tetrahydrocannabinol (THC) are the major neurological and psychiatric cannabinoids, and develop to drugs. It becomes evident that the therapeutic CBD and/or THC are the important candidate of anti-dementia drugs having different mechanism for Alzheimer’s patients. Two receptors and endocannabinoids are also discussed for underlying mechanism of action. In order to promote the breeding of cannabis plant containing higher concentration of target cannabinoid the biosynthetic enzymes were isolated, cloning and the tertiary structure of THCA synthase determined by x-ray analysis resulting in the possibility of molecular breeding for cannabinoids.

Three novel transcription factors involved in cannabinoid biosynthesis in Cannabis sativa L.

Three novel transcription factors were successfully identified and shown to interact with the trichome-specific THCAS promoter regulatory region. Cannabinoids are important secondary metabolites present in Cannabis sativa L. (cannabis). One cannabinoid that has received considerable attention, 9-tetrahydrocannabinol (THC), is derived from Delta-9-Tetrahydrocannabinolic acid (THCA) and responsible for the mood-altering and pain-relieving effects of cannabis. A detailed understanding of transcriptional control of THCA synthase (THCAS) is currently lacking. The primary site of cannabinoid biosynthesis is the glandular trichomes that form on female flowers. Transcription factors (TFs) have been shown to play an important role in secondary-metabolite biosynthesis and glandular trichome formation in Artemisia annua, Solanum lycopersicum and Humulus lupulus. However, analogous information is not available for cannabis. Here, we characterize a 548bp fragment of the THCAS promoter and regulatory region that drives trichome-specific expression. Using this promoter fragment in a yeast-one-hybrid screen, we identified 3 novel TFs (CsAP2L1, CsWRKY1 and CsMYB1) and provided evidence that these 3 TFs regulate the THCAS promoter in planta. The O-Box element within the proximal region of the THCAS promoter is necessary for CsAP2L1-induced transcriptional activation of THCAS promoter. Similar to THCAS, the genes for all three TFs have trichome-specific expression, and subcellular localization of the TFs indicates that all three proteins are in the nucleus. CsAP2L1 and THCAS exhibit a similar temporal, spatial and strain-specific gene expression profiles, while those expression patterns of CsWRKY1 and CsMYB1 are opposite from THCAS. Our results identify CsAP2L1 playing a positive role in the regulation of THCAS expression, while CsWRKY1 and CsMYB1 may serve as negative regulators of THCAS expression.

Systematic review and meta-analysis for the biotechnological production of THC in Morocco

Cannabinoids have promising therapeutic potential. Δ9-tetrahydrocannabinol (THC), the most important psychotropic active ingredient of Cannabis Sativa L, has been the subject of several chemical, pharmacological and biosynthetic studies. In this context, a meta-analysis of biotechnological processes applied to the production of recombinant cannabinoid THC worldwide was carried out. The objective was to highlight the potential of these processes on the Moroccan variety of Cannabis Sativa. The PubMed, ScienceDirect and Web of Science search motors were used to search for original scientific work presenting biotechnological tools used in the production of THC. The scientific articles exploited are those published before the end of 2020. Succinct analysis of the experimental work performed showed that expression of the gene encoding Cannabis Sativa L. THCA synthase was performed on prokaryotic and eukaryotic expression systems. Currently, no functional expression could be obtained in E. coli. Whereas, production of recombinant protein (THCA Synthase) associated with significant enzymatic activity was obtained in P. pastoris cultures (F. Saccharomycetaceae). The exploitation of the sequences showed the presence of a large similarity between the THCA Synthase gene of cannabis Sativa of the Moroccan variety and the mRNA precursor of the same gene reported in several studies. This will allow us to use a specific signal sequence of choice for an adopted expression host, in order to produce the recombinant THCA synthase enzyme from the Moroccan Cannabis Sativa L strain on P. pastoris cultures.

DNA Markers to Discriminate Cannabis sativa L. 'Cheungsam' with Low Tetrahydrocannabinol (THC) Content from Other South Korea Cultivars Based on the Nucleotide Sequences of Tetrahydrocannabinolic Acid Synthase and Putative 3-Ketoacyl-CoA Synthase Genes.

Cannabis sativa L. has been utilized for a long time as a traditional herbal medicine in Korea. Dry fruits, achenes, each containing a single seed of Cannabis, are currently prescribed as Ma In (Cannabis Semen), a laxative. As each achene is enclosed by a bract, in which tetrahydrocannabinol (THC), the main psychological active compound in Cannabis is synthesized; achene is easily contaminated by THC from bract remnants. Therefore, it is safer to harvest achenes from Cannabis with a low THC content. Seeds of hemp, a low THC Cannabis, were recently classified as possible sources of new pharmacologically active compounds. Thus, a proper method to select appropriate Cannabis plants with low THC among cultivars in South Korea for medicinal purpose is necessary. As a result of cross-selection, Cannabis L. cultivar “Cheungsam” (CH) with the lowest THC content among cultivars cultivated in South Korea has been developed. In this study, we developed two DNA markers to reliably discriminate CH from other local cultivars with higher THC contents. We developed primer sets CHF3/CHR2 to amplify the 642 bp DNA marker of CH based on differences in the nucleotide sequences of the THCA synthase gene, which encodes a key enzyme in THC synthesis. We then developed a CHF1/CHR3 primer set to amplify the 401 bp DNA marker of CH based on the differences in both the content of very long chain fatty acids (VLCFs) and the sequence of the putative 3-ketoacyl-CoA synthase (KCS) gene encoding enzymes synthesizing VLCFs among local cultivars.

Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativaL.

Cannabinoids are secondary natural products from the plant Cannabis sativaL. Therapeutic indications of cannabinoids currently comprise a significant area of medicinal research. We have expressed the Δ9-tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) recombinantly in Komagataella phaffii and could detect eight different products with a cannabinoid scaffold after conversion of the precursor cannabigerolic acid (CBGA). Besides five products remaining to be identified, both enzymes were forming three major cannabinoids of C. sativa - Δ9-tetrahydrocannabinolic acid (THCA), cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA). In pursuit of improved enzyme properties for a biotechnological cannabinoid production, we performed site-directed mutagenesis to investigate the glycosylation pattern, the C-terminal berberine-bridge-enzyme (BBE) domain, the active site and the product specificity of both enzymes. The THCAS variant T_N89Q+N499Q (lacking two glycosylation sites) exerted about two-fold increased activity compared to wild-type enzyme. Variant T_H494C+R532C (additional disulfide bridge) exerted about 1.7-fold increased activity compared to wild-type enzyme and a shifted temperature optimum from 52 °C to 57 °C. We generated two CBDAS variants, C_S116A and C_A414V, with 2.8 and 3.3-fold increased catalytic activities for CBDA production. C_A414V additionally showed a broadened pH spectrum and a 19-fold increased catalytic activity for THCA production. These studies lay the groundwork for further research as well as biotechnological cannabinoid production.

Commentary on “Complex Variability within the THCA and CBDA Synthase Genes in Cannabis Species”

Commentary on “Complex Variability within the THCA and CBDA Synthase Genes in Cannabis Species”

Identification of Cannabis sativa L. using the 1-kbTHCA synthase-fluorescence in situ hybridization probe

This study reports a successful application of fluorescence in situ hybridization (FISH) technique in the identification of Cannabis sativa L. cells recovered from fresh and dried powdered plant materials. Two biotin-16-dUTP-labeled FISH probes were designed from the Cannabis-specific tetrahydrocannabinolic acid synthase (THCAS) gene and the ITS region of the 45S rRNA gene. Specificity of probe-target hybridization was tested against the target and 4 non-target plant species, i.e., Humulus lupulus, Mitragyna speciosa, Papaver sp., and Nicotiana tabacum. The 1-kb THCA synthase hybridization probe gave Cannabis-specific hybridization signals, unlike the 700-bp Cannabis-ITS hybridization probe. Probe-target hybridization was also confirmed against 20 individual Cannabis plant samples. The 1-kb THCA synthase and 700-bp Cannabis-ITS hybridization probes clearly showed 2 hybridization signals per cell with reproducibility. The 1-kb THCA synthase probe did not give any FISH signal when tested against H. lupulus, its closely related member of the Canabaceae family. It was also showed that 1-kb THCA synthase FISH probe can be applied to identify small amount of dried powdered Cannabis material with an addition of rehydration step prior to the experimental process. This study provided an alternative identification method for Cannabis trace.

Complex Variability within the THCA and CBDA Synthase Genes in Cannabis Species

Complex Variability within the THCA and CBDA Synthase Genes in Cannabis Species

A PCR marker Linked to a THCA synthase Polymorphism is a Reliable Tool to Discriminate Potentially THC‐Rich Plants of Cannabis sativa L.

Neither absolute THC content nor morphology allows the unequivocal discrimination of fiber cultivars and drug strains of Cannabis sativa L. unequivocally. However, the CBD/THC ratio remains constant throughout the plant's life cycle, is independent of environmental factors, and considered to be controlled by a single locus (B) with two codominant alleles (B(T) and B(D)). The homozygous B(T)/B(T) genotype underlies the THC-predominant phenotype, B(D)/B(D) is CBD predominant, and an intermediate phenotype is induced by the heterozygous state (B(T)/B(D)). Using PCR-based markers in two segregating populations, we proved that the THCA synthase gene represents the postulated B locus and that specific sequence polymorphisms are absolutely linked either to the THC-predominant or the THC-intermediate chemotype. The absolute linkage provides an excellent reliability of the marker signal in forensic casework. For validation, the species-specific marker system was applied to a large number of casework samples and fiber hemp cultivars.

Analysis of THCA synthase gene expression in cannabis: A preliminary study by real-time quantitative PCR

In this paper we describe analyses performed by Real-Time Reverse-Transcriptase Polymerase Chain Reaction (real-time RT-PCR) on RNA of 12 samples, carried out for forensic purposes to investigate a correlation between tetrahydrocannabinol (THC) concentration in Cannabis and the tetrahydrocannabinol acid synthase (THCAS) gene expression.Samples were obtained from an experimental cultivation of declared potency Cannabis variety seeds and from seizures. The Rubisco gene and the 26S ribosomal RNA gene were used as internal control genes for their constant expression and stability.As results we found minor gene expression in samples from leaves of young plants.Further, grouping results for cannabis samples with similar characteristics, we have found an increased relative expression in samples with the highest percentage of THC coming from seized sample and adult plants.

Structure and Function of ∆1-Tetrahydrocannabinolic Acid (THCA) Synthase, the Enzyme Controlling the Psychoactivity of Cannabis sativa

∆1-Tetrahydrocannabinolic acid (THCA) synthase catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) into THCA, the precursor of the primary psychoactive agent ∆1-tetrahydrocannabinol in Cannabis sativa. The enzyme was overproduced in insect cells, purified, and crystallized in order to investigate the structure-function relationship of THCA synthase, and the tertiary structure was determined to 2.75Å resolution by X-ray crystallography (R(cryst)=19.9%). The THCA synthase enzyme is a member of the p-cresol methyl-hydroxylase superfamily, and the tertiary structure is divided into two domains (domains I and II), with a flavin adenine dinucleotide coenzyme positioned between each domain and covalently bound to His114 and Cys176 (located in domain I). The catalysis of THCA synthesis involves a hydride transfer from C3 of CBGA to N5 of flavin adenine dinucleotide and the deprotonation of O6' of CBGA. The ionized residues in the active site of THCA synthase were investigated by mutational analysis and X-ray structure. Mutational analysis indicates that the reaction does not involve the carboxyl group of Glu442 that was identified as the catalytic base in the related berberine bridge enzyme but instead involves the hydroxyl group of Tyr484. Mutations at the active-site residues His292 and Tyr417 resulted in a decrease in, but not elimination of, the enzymatic activity of THCA synthase, suggesting a key role for these residues in substrate binding and not direct catalysis.

Discrimination of ‘fiber-type’ and ‘drug-type’ Cannabis sativa L. by fluorescent duplex PCR

A fluorescent duplex-PCR test was developed based on polymorphisms of the THCA synthase gene in order to discriminate the fiber- and drug-type Cannabis sativa L. and to indicate the presence of Cannabis trace in suspected materials by the numbers and sizes of PCR-amplified products. DNA analysis of drug-type Cannabis resulted in two different PCR-amplified DNA fragments of 94 and 158 bp, whereas only the 94 bp PCR product was amplified from the fiber-type DNA. DNA test results of another 6 Cannabis sativa L. collected from the field agreed with chemotype determined by GC-MS. However, it was noted that the only intermediate drug-type sample tested gave a drug-type result for DNA testing. Specificity of the duplex PCR was shown by testing with DNA from species that may be related to Cannabis abuse, i.e., common hop ( Humulus lupulus L.), 2 narcotic plants ( Papaver somniferum and Mitragyna speciosa), tobacco ( Nicotiana tabacum) and human. Sensitivity of detection was as low as 100 pg of genomic DNA.

Production of Δ 1-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris

Δ 1-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Δ 1-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted ∼1.32 nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a ∼98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure–function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.

Crystallization of Δ1-tetrahydrocannabinolic acid (THCA) synthase fromCannabis sativa

Delta1-Tetrahydrocannabinolic acid (THCA) synthase is a novel oxidoreductase that catalyzes the biosynthesis of the psychoactive compound THCA in Cannabis sativa (Mexican strain). In order to investigate the structure-function relationship of THCA synthase, this enzyme was overproduced in insect cells, purified and finally crystallized in 0.1 M HEPES buffer pH 7.5 containing 1.4 M sodium citrate. A single crystal suitable for X-ray diffraction measurement was obtained in 0.09 M HEPES buffer pH 7.5 containing 1.26 M sodium citrate. The crystal diffracted to 2.7 A resolution at beamline BL41XU, SPring-8. The crystal belonged to the primitive cubic space group P432, with unit-cell parameters a = b = c = 178.2 A. The calculated Matthews coefficient was approximately 4.1 or 2.0 A3 Da(-1) assuming the presence of one or two molecules of THCA synthase in the asymmetric unit, respectively.

ChemInform Abstract: Pharmacognosical Study on Secondary Metabolites

Clonal micropropagation on various medicinal plants was set up resulting in the regenerated plants which possessed a homogeneous quality. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization of mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigene-BSA conjugate with mouse myeloma cells. Competitive enzyme-linked immunosorbent assay (ELISA) using MAb was set up as a high sensitive, specific and reproducible qualitative method. A method of determination for ginsenosides by using a unique western blotting was established. Immunoaffinity column chromatography using an anti-ginsenoside Rb1MAb has made possible a single-step separation of ginsenoside Rb1 from a crude ginseng extract. Single chain Fv gene of anti-forskolin MAb was prepared from mRNA of hybridoma secreting anti-forskolin MAb and cloned. Gene was constructed into a pET-28a(+) vector producing a scFv protein. Modeling of forskolin and scFV was investigated. THCA synthase was purified from the homogenate of Cannabis sativa leaves on successive column chromatographies. THCA synthase was confirmed to be homogeneity having 75 kDa. To obtain the corresponding cDNA clone of THCA synthase, a set of degenerate promers was constructed based on N-terinal and internal amino acid sequences of THCA synthase. The 5' and 3' ends of cDNA were amplified by RACE. A full sequencing has been determined to be corded a polypeptide having 545 amino acid residues. The cDNA clone was expressed in yeast system via PUC19 vector resulting in THCA synthase activity.

植物二次代謝産物に関わる生薬学的研究

Clonal micropropagation on various medicinal plants was set up resulting in the regenerated plants which possessed a homogeneous quality. The ratio of hapten to bovine serum albumin (BSA) in an antigen conjugate was determined by matrix-assisted laser desorption/ionization of mass spectrometry. A hybridoma secreting monoclonal antibody (MAb) was produced by fusing splenocytes immunized with an antigene-BSA conjugate with mouse myeloma cells. Competitive enzyme-linked immunosorbent assay (ELISA) using MAb was set up as a high sensitive, specific and reproducible qualitative method. A method of determination for ginsenosides by using a unique western blotting was established. Immunoaffinity column chromatography using an anti-ginsenoside Rb1MAb has made possible a single-step separation of ginsenoside Rb1 from a crude ginseng extract. Single chain Fv gene of anti-forskolin MAb was prepared from mRNA of hybridoma secreting anti-forskolin MAb and cloned. Gene was constructed into a pET-28a(+) vector producing a scFv protein. Modeling of forskolin and scFV was investigated. THCA synthase was purified from the homogenate of Cannabis sativa leaves on successive column chromatographies. THCA synthase was confirmed to be homogeneity having 75 kDa. To obtain the corresponding cDNA clone of THCA synthase, a set of degenerate promers was constructed based on N-terinal and internal amino acid sequences of THCA synthase. The 5' and 3' ends of cDNA were amplified by RACE. A full sequencing has been determined to be corded a polypeptide having 545 amino acid residues. The cDNA clone was expressed in yeast system via PUC19 vector resulting in THCA synthase activity.