Cannabidiolic Acid Synthase Research Articles

Low UV radiation influenced DNA methylation, gene regulation, cell proliferation, viability, and biochemical differentiation in the cell suspension cultures of Cannabis indica

The effect of low artificial Ultraviolet (UV) on the DNA methylation remains controversial. This study addresses how differential photoperiods of UV radiation affect the biochemical and molecular behaviors of Cannabis indica cell suspension cultures. The cell suspensions were illuminated with the compact fluorescent lamps (CFL), emitting a combination of 10% UVB, 30% UVA, and the rest visible wavelengths for 0, 4, 8, and 16 h. The applied photoperiods influenced cell morphological characteristics. The 4 h photoperiod was the most effective treatment for improving biomass, growth index and cell viability percentage while these indices remained non-significant in the 16 h treatment. The methylation-sensitive amplified polymorphism (MASP) assay revealed that the UV radiation was epigenetically accompanied by DNA hypermethylation. The light-treated cells significantly displayed higher relative expression of the cannabidiolic‌ acid synthase (CBDAS) and delta9-tetrahydrocannabinolic acid synthase (THCAS) genes about 4-fold. The expression of the olivetolic acid cyclase (OAC) and olivetol synthase (OLS) genes exhibited an upward trend in response to the UV radiation. The light treatments also enhanced the proline content and protein concentration. The 4 h illumination was significantly capable of improving the cannabidiol (CBD) and delta-9-tetrahydrocannabinol (THC) concentrations, in contrast with 16 h. By increasing the illumination exposure time, the activity of the phenylalanine ammonia-lyase (PAL) enzyme linearly upregulated. The highest amounts of the phenylpropanoid derivatives were observed in the cells cultured under the radiation for 4 h. Taken collective, artificial UV radiation can induce DNA methylation modifications and impact biochemical and molecular differentiation in the cell suspensions in a photoperiod-dependent manner.

The development of a next-generation sequencing panel targeting cannabinoid synthase genes to distinguish between marijuana and hemp.

Hemp and marijuana, both derived from Cannabis sativa L. (C. sativa), are subject to divergent legal regulations due to their different Δ9-tetrahydrocannabinol (Δ9-THC) contents. Cannabinoid synthase genes are considered the key enzymes that determine the chemical composition or chemotype of a particular cultivar. However, existing methods for crop type differentiation based on previous synthase gene theories have limitations in terms of precision and specificity, and a wider range of cannabis varieties must be considered when examining cannabis-based genetic markers. A custom next-generation sequencing (NGS) panel was developed targeting all synthase genes, including Δ9-THC acid synthase, cannabidiolic acid synthase, and cannabichromenic acid synthase, as well as the pseudogenes across diverse C. sativa samples, spanning reference hemp and marijuana, commercial hemp derivatives, and seized marijuana extracts. Interpretation of NGS data revealed a relationship between genotypes and underlying chemotypes, with the principal component analysis indicating a clear distinction between hemp and marijuana clusters. This differentiation was attributed to variations in both synthase genes and pseudogene variants. Finally, this study proposes a genetic cannabis classification method using a differentiation flow chart with novel synthase markers. The flow chart successfully differentiated hemp from marijuana with a 1.3% error rate (n=147).

Improvement of cannabidiolic acid synthetase activity through molecular docking and site-directed mutagenesis

Cannabidiol (CBD), a nonhallucinogenic but therapeutic compound, is widely used in fields such as medicine, food, health products, and cosmetics. Cannabidiolic acid synthase (CBDAS) can exclusively catalyze the conversion of cannabigerolic acid (CBGA), which is the common precursor of cannabinoids, to CBDA, which is then decarboxylated to form CBD. In order to determine which amino acids in the CBDAS protein undergo mutations that can significantly enhance the catalytic activity of the enzyme, molecular docking and experimental validation were performed in this study. The CBDAS gene was cloned from a high-CBD-content hemp germplasm; this 1635 base pair gene encodes 544 amino acids. Saturation and alanine scanning mutagenesis and combinatorial mutation analysis showed that a single amino acid mutation (C176W) and three dual mutations (H69G+H114L, H114L+C176W and G183V+N482W) in CBDAS reduced the binding energy of the mutant for the substrate CBGA. The molecular docking results indicated that for the mutants CBDASH114L+C176W and CBDASG183V+N482W, the interaction force with CBGA was increased, the bond distance was shortened, and some amino acids that interacted with the substrate were also altered compared with the WT. For the mutants CBDASC176W and CBDASH69G+H114L, compared with the WT, the interaction force with CBGA was decreased, or the interaction was eliminated. The results of enzyme catalytic activity measurement showed that compared with the WT, the catalytic activities of CBDASG183V+N482W and CBDASH114L+C176W were significantly increased, and that of CBDASH69G+H114L was significantly decreased (P < 0.05), which was generally consistent with the expected results from molecular docking. The crude leaf extract, when used as the substrate, produced more CBDA and CBD than an equal concentration of the CBGA standard. Obviously, it is possible that the recombinant CBDAS produced with yeast mutants can be used for secondary production of CBD in the process of cannabinoid extraction with the remaining CBGA in the leaves as the substrate. The research results provide a theoretical basis for the molecular modification of other cannabinoid synthetases.

Exogenous application of stress-related signaling molecules affect growth and cannabinoid accumulation in medical cannabis (Cannabis sativa L.).

Medical cannabis (Cannabis sativa L.) is a source of bioactive phytochemicals with promising pharmacological and therapeutic applications. Enhancing the accumulation of valuable bioactive compounds is potentially a way of increasing the economic importance of this crop. Signaling molecules like salicylic acid (SA), jasmonic acid (JA), and γ-aminobutyric acid (GABA) are involved in the regulation of plant development and responses to biotic and abiotic stresses. Moreover, several phytohormones regulate plant trichome formation and elicit the synthesis of secondary metabolites in many plant species in both in vitro and in vivo systems. Therefore, exogenously delivered plant signaling molecules have the potential to modify the chemical profiles of medical cannabis. In this study, we found that the foliar application of SA, methyl jasmonate (MeJA), and GABA produces changes in the accumulation of the two major cannabinoids, cannabidiolic acid (CBDA) and Δ9- tetrahydrocannabinolic acid (THCA), in leaves and inflorescences of a medical cannabis variety. MeJA at 0.1 mM increased the CBDA content in inflorescences by 15.6%, while SA and MeJA at 0.1 mM increased CBDA and THCA accumulation in leaves by up to 57.3%. Treatments did not change the expression of genes participating in the final steps of the biosynthetic pathway of cannabinoids: olivetolic acid cyclase (CsOAC-1 and CsOAC-2), 2-acylphloroglucinol 4-prenyltransferase (CsPT4), cannabidiolic acid synthase (CsCBDAS), and tetrahydrocannabinolic acid synthase (CsTHCAS). Trichome density was not significantly different from the control plants in any treatment. Besides, we found strong correlations between several plant growth parameters and cannabinoid yields, showing a direct link between plant fitness and the production of cannabinoids.

Simulated microgravity contributed to modification of callogenesis performance and secondary metabolite production in CannabisIndica

In vitro plant culture paves the way for meeting the industrial demand of pharmaceutically valuable secondary metabolites. This study intends to monitor how callus cells of Cannabis indica respond to the simulated microgravity (clinorotation; a Man-made technology). Callus initiation resulted from the culture of the leaf explant in a medium supplemented with kinetin (0.5 mgL−1) and 2, 4-D (2 mgL−1). Calli were treated with microgravity at three exposure times (0, 3, and 5 days). The microgravity treatments increased callus biomass about 2.5-fold. The clinorotation treatments transcriptionally induced the olivetolic acid cyclase (OAC) and olivetol synthase (OLS) genes about 6.2-fold. The tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes displayed a similar upward trend in response to microgravity. The applied treatments also stimulated the expression of the ethylene-responsive element-binding proteins (ERF1B) and WRKY1 transcription factors by an average of 7.6-fold. Moreover, the simulated microgravity triggered epigenetic modification in the DNA methylation profile. The HPLC-based assessment validated the high efficacy of the clinorotation treatments to increase the concentration of cannabinoids, including Cannabigerol (CBG) and Cannabidiol (CBD). However, the clinorotated calli contained a lower concentration of Tetrahydrocannabinol (THC) than the control group. The microgravity treatments increased concentrations of proline (79%), soluble sugars (61.3%), and proteins (21.4%) in calli. The biochemical assessment revealed that the clinorotation treatments slightly increased H2O2 concentration. The upregulation in the activities of peroxidase, catalase, and phenylalanine ammonia-lyase enzymes resulted from the microgravity treatments. Both HPLC and molecular assessments validated the significant efficacy of microgravity to enhance the production of cannabinoids.

Analysis of Sequence Variability and Transcriptional Profile of Cannabinoid synthase Genes in Cannabis sativa L. Chemotypes with a Focus on Cannabichromenic acid synthase.

Cannabis sativa L. has been long cultivated for its narcotic potential due to the accumulation of tetrahydrocannabinolic acid (THCA) in female inflorescences, but nowadays its production for fiber, seeds, edible oil and bioactive compounds has spread throughout the world. However, some hemp varieties still accumulate traces of residual THCA close to the 0.20% limit set by European Union, despite the functional gene encoding for THCA synthase (THCAS) is lacking. Even if some hypotheses have been produced, studies are often in disagreement especially on the role of the cannabichromenic acid synthase (CBCAS). In this work a set of European Cannabis genotypes, representative of all chemotypes, were investigated from a chemical and molecular point of view. Highly specific primer pairs were developed to allow an accurate distinction of different cannabinoid synthases genes. In addition to their use as markers to detect the presence of CBCAS at genomic level, they allowed the analysis of transcriptional profiles in hemp or marijuana plants. While the high level of transcription of THCAS and cannabidiolic acid synthase (CBDAS) clearly reflects the chemical phenotype of the plants, the low but stable transcriptional level of CBCAS in all genotypes suggests that these genes are active and might contribute to the final amount of cannabinoids.

Identification of cis conformation natural rubber and proteins in Ficus altissima Blume latex

Ficus altissima Blume, a horticultural plant in tropical and warm subtropical regions, can produce natural rubber with comparable molecular weight to the Hevea brasiliensis rubber. The F. altissima latex has an acidic pH (about 4.89). The rubber particle size distribution is a unimodal profile, and the peak frequency is at a size of 4.5 μm. The natural rubber of F. altissima was determined to be a cis conformation via 13C NMR. The Mp (molecular weight of the peak maxima) of the deproteinized F. altissima rubber was 9.34 × 105 Da. LC-MS was used to identify the proteins of rubber particles and serum. The most abundant protein of the creamy rubber particle layer is an acid phosphatase, while the most abundant proteins of serum were an (R)-mandelonitrilelyase and a polygalacturonase inhibitor. Pharmaceutical proteins (ficins) or enzymes related to the biosynthesis of natural medicines (a cannabidiolic acid synthase and two lupeol synthase) were identified in F. altissima latex. The data of this study may be helpful for research on the functions of latex in latex-borne plants and the biosynthesis mechanism of natural rubber.

A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana.

Summary Demand for cannabidiol (CBD), the predominant cannabinoid in hemp (Cannabis sativa), has favored cultivars producing unprecedented quantities of CBD. We investigated the ancestry of a new cultivar and cannabinoid synthase genes in relation to cannabinoid inheritance.A nanopore‐based assembly anchored to a high‐resolution linkage map provided a chromosome‐resolved genome for CBDRx, a potent CBD‐type cultivar. We measured cannabinoid synthase expression by cDNA sequencing and conducted a population genetic analysis of diverse Cannabis accessions. Quantitative trait locus mapping of cannabinoids in a hemp × marijuana segregating population was also performed.Cannabinoid synthase paralogs are arranged in tandem arrays embedded in long terminal repeat retrotransposons on chromosome 7. Although CBDRx is predominantly of marijuana ancestry, the genome has cannabidiolic acid synthase (CBDAS) introgressed from hemp and lacks a complete sequence for tetrahydrocannabinolic acid synthase (THCAS). Three additional genomes, including one with complete THCAS, confirmed this genomic structure. Only cannabidiolic acid synthase (CBDAS) was expressed in CBD‐type Cannabis, while both CBDAS and THCAS were expressed in a cultivar with an intermediate tetrahydrocannabinol (THC) : CBD ratio.Although variation among cannabinoid synthase loci might affect the THC : CBD ratio, variability among cultivars in overall cannabinoid content (potency) was also associated with other chromosomes.

A draft phased assembly of the diploid Cascade hop (Humulus lupulus) genome.

Hop (Humulus lupulus L. var Lupulus) is a diploid, dioecious plant with a history of cultivation spanning more than one thousand years. Hop cones are valued for their use in brewing and contain compounds of therapeutic interest including xanthohumol. Efforts to determine how biochemical pathways responsible for desirable traits are regulated have been challenged by the large (2.8 Gb), repetitive, and heterozygous genome of hop. We present a draft haplotype-phased assembly of the Cascade cultivar genome. Our draft assembly and annotation of the Cascade genome is the most extensive representation of the hop genome to date. PacBio long-read sequences from hop were assembled with FALCON and partially phased with FALCON-Unzip. Comparative analysis of haplotype sequences provides insight into selective pressures that have driven evolution in hop. We discovered genes with greater sequence divergence enriched for stress-response, growth, and flowering functions in the draft phased assembly. With improved resolution of long terminal retrotransposons (LTRs) due to long-read sequencing, we found that hop is over 70% repetitive. We identified a homolog of cannabidiolic acid synthase (CBDAS) that is expressed in multiple tissues. The approaches we developed to analyze the draft phased assembly serve to deepen our understanding of the genomic landscape of hop and may have broader applicability to the study of other large, complex genomes.

A comprehensive molecular approach to the detection of drug-type versus fiber-type hemp varieties

The availability of molecular markers able to distinguish drug-type from fiber-type Cannabis sativa cultivars would allow fast and cheap analysis of any plant specimen, including seeds and leaves. Several approaches to this issue have been described, mainly using polymorphisms in the genes coding for tetrahydrocannabinol acid synthase or cannabidiolic acid synthase. Some studies reported sequencing of these genes from small groups of hemp varieties belonging to both chemotypes, showing the occurrence of specific DNA signatures. However, the effectiveness of the corresponding primers to discriminate among chemotypes has been validated on a limited number of cultivars, or not tested at all. Here we report a thorough in silico analysis of available gene sequences for both synthases, showing the existence of hypervariable regions at 3’ and 5’ ends. This notwithstanding, some possible signatures were identified, and 12 putatively specific primer pairs were designed and tested on 16 fiber-type and 11 drug-type varieties. In most cases inconsistent results were obtained, further strengthening the high genetic variability of these genes in hemp germplasm, yet some highly informative polymorphisms were identified. Potentiality and perspectives of this approach are discussed.

Rapid identification of drug-type and fiber-type cannabis by allele specific duplex PCR

Cannabis is classified into two types: drug-type cannabis, which is abused worldwide, and fiber-type cannabis, which is used for industrial purposes. The two types are a result of differences in the sequences of tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes. In the present study, we aimed to establish a PCR-based method to distinguish between drug-type and fiber-type cannabis by detecting the differences in the sequences of THCAS and CBDAS. We constructed a single-plex PCR targeting active THCAS, and observed drug-type cannabis-specific amplification when using 10pg to 1ng of DNA; however, amplification was also observed in fiber-type cannabis when the DNA content reached 10ng. Similarly, single-plex PCR targeting active CBDAS showed fiber-type cannabis-specific amplification in 100pg of DNA, as well as in >1ng of drug-type cannabis DNA. Therefore, when an allele-specific duplex PCR system was constructed, in which both primer sets were mixed at an appropriate ratio, unintended nonspecific amplification was suppressed and amplicons of different sizes were observed between the drug-type and fiber-type cannabis, using DNA samples in the range of 1pg to 10ng. When the constructed duplex PCR was performed on DNA extracted from various cannabis seed samples, it was possible to distinguish between the drug-type and the fiber-type as well as detect a hybrid-type with both active THCAS and active CBDAS and a special type with neither. The identification method developed in the present study can quickly and accurately distinguish between drug-type and fiber-type cannabis, and is expected to be used for various purposes such as the detection of genetic contamination of industrial hemp as well as forensic examination of cannabis-related cases.

Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa.

PremiseHow genetic variation within a species affects phytochemical composition is a fundamental question in botany. The ratio of two specialized metabolites in Cannabis sativa, tetrahydrocannabinol (THC) and cannabidiol (CBD), can be grouped into three main classes (THC‐type, CBD‐type, and intermediate type). We tested a genetic model associating these three groups with functional and nonfunctional alleles of the cannabidiolic acid synthase gene (CBDAS).MethodsWe characterized cannabinoid content and assayed CBDAS genotypes of >300 feral C. sativa plants in Minnesota, United States. We performed a test cross to assess CBDAS inheritance. Twenty clinical cultivars obtained blindly from the National Institute on Drug Abuse and 12 Canadian‐certified grain cultivars were also examined.ResultsFrequencies of CBD‐type, intermediate‐type, and THC‐type feral plants were 0.88, 0.11, and 0.01, respectively. Although total cannabinoid content varied substantially, the three groupings were perfectly correlated with CBDAS genotypes. Genotype frequencies observed in the test cross were consistent with codominant Mendelian inheritance of the THC:CBD ratio. Despite significant mean differences in total cannabinoid content, CBDAS genotypes blindly predicted the THC:CBD ratio among clinical cultivars, and the same was true for industrial grain cultivars when plants exhibited >0.5% total cannabinoid content.ConclusionsOur results extend the generality of the inheritance model for THC:CBD to diverse C. sativa accessions and demonstrate that CBDAS genotyping can predict the ratio in a variety of practical applications. Cannabinoid profiles and associated CBDAS segregation patterns suggest that feral C. sativa populations are potentially valuable experimental systems and sources of germplasm.

Cold Plasma Up-Regulated Expressions of WRKY1 Transcription Factor and Genes Involved in Biosynthesis of Cannabinoids in Hemp (Cannabis sativa L.)

Plasma technology as an eco-friendly efficient strategy has gained much attention in various industries, especially in food, medicine, and agriculture. This study aimed to explore the cold plasma-mediated changes in growth, anatomy, expression of a WRKY1 transcription factor, and transcription rates of four key genes involved in the biosynthesis of cannabinoids (pharmaceutically valuable secondary metabolites) in hemp (Cannabis sativa L.). The seeds were treated with cold plasma (dielectric barrier discharge; 0.84 W cm−2; exposure times of 0, 40, and 80 s). The plasma treatment of 40 s increased biomass in both shoot and roots by an average of 57%, whereas the treatment at 80 s delayed growth and reduced it by 48%. Seed priming with plasma up-regulated the WRKY1 transcription factor (mean = 11.55 folds). Besides, the plasma treatments induced the expression of olivetolic acid cyclase by 42 folds. Furthermore, the plasma-primed seedlings also exhibited higher expression rates of olivetol synthase by 19 folds. With a similar trend, exposure to plasma stimulated transcription of cannabidiolic acid synthase by 12.4 folds. Up-regulations in Δ9-tetrahydrocannabinolic acid synthase also occurred following seed priming with plasma by 25.6 folds. Seed priming with plasma exhibits high potency to up-regulate expressions of genes involved in the productions of secondary metabolites, like cannabinoids. These results imply that the plasma reception and signal transduction can alter expressions of genes at the transcriptional level through which plasma priming may improve plant protection and secondary metabolism.

Highly Predictive Genetic Markers Distinguish Drug-Type from Fiber-Type Cannabis sativa L.

Genetic markers can be used in seeds and in plants to distinguish drug-type from fiber-type Cannabis Sativa L. varieties even at early stages, including pre-germination when cannabinoids are not accumulated yet. With this aim, this paper reports sequencing results for tetrahydrocannabinolic acid synthase (THCAS) and cannabidiolic acid synthase (CBDAS) genes from 21 C. sativa L. varieties. Taking into account that THCAS- and CBDAS-derived enzymes compete for the same substrate, the novelty of this work relies in the identification of markers based on both THCAS and CBDAS rather than THCAS alone. Notably, in our panel, we achieved an adequate degree of discrimination (AUC 100%) between drug-type and fiber-type cannabis samples. Our sequencing approach allowed identifying multiple genetic markers (single-nucleotide polymorphisms—SNPs—and a deletion/insertion) that effectively discriminate between the two subgroups of cannabis, namely fiber type vs. drug type. We identified four functional SNPs that are likely to induce decreased THCAS activity in the fiber-type cannabis plants. We also report the finding on a deletion in the CBDAS gene sequence that produces a truncated protein, possibly resulting in loss of function of the enzyme in the drug-type varieties. Chemical analyses for the actual concentration of cannabinoids confirmed the identification of drug-type rather than fiber-type genotypes. Genetic markers permit an early identification process for forensic applications while simplifying the procedures related to detection of therapeutic or industrial hemp.

Enhanced tolerance of industrial hemp (Cannabis sativa L.) plants on abandoned mine land soil leads to overexpression of cannabinoids.

Industrial activities have a detrimental impact on the environment and health when high concentrations of pollutants are released. Phytoremediation is a natural method of utilizing plants to remove contaminants from the soil. The goal of this study was to investigate the ability of Cannabis sativa L. to sustainably grow and remediate abandoned coal mine land soils in Pennsylvania. In this study, six different varieties of industrial hemp (Fedora 17, Felina 32, Ferimon, Futura 75, Santhica 27, and USO 31) were grown on two different contaminated soil types and two commercial soils (Miracle-Gro Potting Mix and PRO-MIX HP Mycorrhizae High Porosity Grower Mix). Plants growing in all soil types were exposed to two environmental conditions (outside and in the greenhouse). Seed germination response and plant height indicated no significant differences among all hemp varieties grown in different soils, however on an average, the height of the plants grown in the greenhouse exceeded that of the plants grown outdoors. In addition, heavy metal analysis of Arsenic, Lead, Nickel, Mercury, and Cadmium was performed. The concentration of Nickel was 2.54 times greater in the leaves of hemp grown in mine land soil outdoors when compared to greenhouse conditions. No differences were found between expression of heavy metal transporter genes. Secondary metabolite analysis of floral buds from hemp grown in mine land soil displayed a significant increase in the total Cannabidiol content (2.16%, 2.58%) when compared to Miracle-Gro control soil (1.08%, 1.6%) for outdoors and in the greenhouse, respectively. Molecular analysis using qRT-PCR indicated an 18-fold increase in the expression of the cannabidiolic acid synthase gene in plants grown on mine land soil. The data indicates a high tolerance to heavy metals as indicated from the physiological and metabolites analysis.

Cannabinoid synthases and osmoprotective metabolites accumulate in the exudates of Cannabis sativa L. glandular trichomes

Cannabinoids are terpenophenolic compounds produced by Cannabis sativa L., which accumulate in storage cavities of glandular trichomes as a part of the exudates. We investigated if tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase, which are involved in the last step of cannabinoid biosynthesis, are also secreted into Cannabis trichome exudates. The exudates were collected by microsuction from storage cavities of Cannabis glandular trichomes and were subjected for proteomic and metabolomic analyses. The catalytic activity of the exudates was documented by cannabigerolic acid biotransformation studies under hydrophobic conditions. Electrophoretic separations revealed protein bands at ˜65 kDa, which were further identified as tetrahydrocannabinolic acid synthase and cannabidiolic acid synthase. The accumulation of the enzymes in trichome exudates increased substantially during the flowering period in the drug-type Cannabis plants. The content of cannabinoids increased significantly after incubating hexane-diluted trichome exudates with cannabigerolic acid. In this study, we showed that Cannabis glandular trichomes secrete and accumulate cannabinoid synthases in storage cavities, and the enzymes able to convert cannabigerolic acid under hydrophobic trichome-mimicking conditions. Metabolite profiling of the exudates revealed compounds with hydrophilic, osmoprotective and amphiphilic properties, which may play a role in providing a necessary aqueous microenvironment, which enables enzyme solubility and biocatalysis under hydrophobic conditions of glandular trichomes.

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.

Science of Industrial Hemp: On the Leading Edge of a Growing Industry

Science of Industrial Hemp: On the Leading Edge of a Growing Industry

Gene duplication and divergence affecting drug content in Cannabis sativa.

Cannabis sativa is an economically important source of durable fibers, nutritious seeds, andpsychoactive drugs but few economic plants are so poorly understood genetically. Marijuana and hemp were crossed to evaluate competing models of cannabinoid inheritance and to explain the predominance of tetrahydrocannabinolic acid (THCA) in marijuana compared with cannabidiolic acid (CBDA) in hemp. Individuals in the resulting F2 population were assessed for differential expression of cannabinoid synthase genes and were used in linkage mapping. Genetic markers associated with divergent cannabinoid phenotypes were identified. Although phenotypic segregation and a major quantitative trait locus (QTL) for the THCA/CBDA ratio were consistent with a simple model of codominant alleles at a single locus, the diversity of THCA and CBDA synthase sequences observed in the mapping population, the position of enzyme coding loci on the map, and patterns of expression suggest multiple linked loci. Phylogenetic analysis further suggests a history of duplication and divergence affecting drug content. Marijuana is distinguished from hemp by a nonfunctional CBDA synthase that appears to have been positively selected to enhance psychoactivity. An unlinked QTL for cannabinoid quantity may also have played a role in the recent escalation of drug potency.

The draft genome and transcriptome of Cannabis sativa

BackgroundCannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored.ResultsWe sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of Δ9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp.ConclusionsThe availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.