Engineering cannabinoid production in Saccharomyces cerevisiae.

Phytocannabinoids are natural products with highly interesting pharmacological properties mainly produced by plants. The production of cannabinoids in a heterologous host system has gained interest in recent years as a promising alternative to production from plant material. However, the systems reported so far do not achieve industrially relevant titers, highlighting the need for alternative systems. Here, we show the production of the cannabinoids cannabigerolic acid and cannabigerol from glucose and hexanoic acid in a heterologous yeast system using the aromatic prenyltransferase NphB from Streptomyces sp. strain CL190. The production was significantly increased by introducing a fusion protein consisting of ERG20WW and NphB. Furthermore, we improved the production of the precursor olivetolic acid to a titer of 56 mg L-1 . The implementation of the cannabinoid synthase genes enabled the production of Δ9 -tetrahydrocannabinolic acid, cannabidiolic acid as well as cannabichromenic acid, where the heterologous biosynthesis of cannabichromenic acid in a yeast system was demonstrated for the first time. In addition, we found that the product spectrum of the cannabinoid synthases localized to the vacuoles of the yeast cells was highly dependent on extracellular pH, allowing for easy manipulation. Finally, using a fed-batch approach, we showed cannabigerolic acid and olivetolic acid titers of up to 18.2 mg L-1 and 117 mg L-1 , respectively.

Bioengineering of the Marine Diatom Phaeodactylum tricornutum with Cannabis Genes Enables the Production of the Cannabinoid Precursor, Olivetolic Acid.

The increasing demand for novel natural compounds has prompted the exploration of innovative approaches in bioengineering. This study investigates the bioengineering potential of the marine diatom Phaeodactylum tricornutum through the introduction of cannabis genes, specifically, tetraketide synthase (TKS), and olivetolic acid cyclase (OAC), for the production of the cannabinoid precursor, olivetolic acid (OA). P. tricornutum is a promising biotechnological platform due to its fast growth rate, amenability to genetic manipulation, and ability to produce valuable compounds. Through genetic engineering techniques, we successfully integrated the cannabis genes TKS and OAC into the diatom. P. tricornutum transconjugants expressing these genes showed the production of the recombinant TKS and OAC enzymes, detected via Western blot analysis, and the production of cannabinoids precursor (OA) detected using the HPLC/UV spectrum when compared to the wild-type strain. Quantitative analysis revealed significant olivetolic acid accumulation (0.6-2.6 mg/L), demonstrating the successful integration and functionality of the heterologous genes. Furthermore, the introduction of TKS and OAC genes led to the synthesis of novel molecules, potentially expanding the repertoire of bioactive compounds accessible through diatom-based biotechnology. This study demonstrates the successful bioengineering of P. tricornutum with cannabis genes, enabling the production of OA as a precursor for cannabinoid production and the synthesis of novel molecules with potential pharmaceutical applications.

Characterization and co-expression analysis of ATP-binding cassette transporters provide insight into genes related to cannabinoid transport in Cannabis sativa L.

Plant ATP-binding cassette (ABC) transporters contribute the transport of diverse secondary metabolites. However, their roles in cannabinoid trafficking are still unsolved in Cannabis sativa. In this study, 113 ABC transporters were identified and characterized in C. sativa from their physicochemical properties, gene structure, and phylogenic relationship, as well as spatial gene expression patterns. Eventually, seven core transporters were proposed including one member in ABC subfamily B (CsABCB8) and six ABCG members (CsABCG4, CsABCG10, CsABCG11, CsABCG32, CsABCG37, and CsABCG41), harboring potential in participating cannabinoid transport, by combining phylogenetic and co-expression analysis from the gene and metabolite level. The candidate genes exhibited a high correlation with cannabinoid biosynthetic pathway genes and the cannabinoid content, and they were highly expressed where cannabinoids appropriately biosynthesized and accumulated. The findings underpin further research on the function of ABC transporters in C. sativa, especially in unveiling the mechanisms of cannabinoid transport to boost systematic and targeted metabolic engineering.

Oxylipin biosynthetic gene families of Cannabis sativa.

Cannabis sativa is a global multi-billion-dollar cash crop with numerous industrial uses, including in medicine and recreation where its value is largely owed to the production of pharmacological and psychoactive metabolites known as cannabinoids. Often underappreciated in this role, the lipoxygenase (LOX)-derived green leaf volatiles (GLVs), also known as the scent of cut grass, are the hypothetical origin of hexanoic acid, the initial substrate for cannabinoid biosynthesis. The LOX pathway is best known as the primary source of plant oxylipins, molecules analogous to the eicosanoids from mammalian systems. These molecules are a group of chemically and functionally diverse fatty acid-derived signals that govern nearly all biological processes including plant defense and development. The interaction between oxylipin and cannabinoid biosynthetic pathways remains to be explored. Despite their unique importance in this crop, there has not been a comprehensive investigation focusing on the genes responsible for oxylipin biosynthesis in any Cannabis species. This study documents the first genome-wide catalogue of the Cannabis sativa oxylipin biosynthetic genes and identified 21 LOX, five allene oxide synthases (AOS), three allene oxide cyclases (AOC), one hydroperoxide lyase (HPL), and five 12-oxo-phytodienoic acid reductases (OPR). Gene collinearity analysis found chromosomal regions containing several isoforms maintained across Cannabis, Arabidopsis, and tomato. Promoter, expression, weighted co-expression genetic network, and functional enrichment analysis provide evidence of tissue- and cultivar-specific transcription and roles for distinct isoforms in oxylipin and cannabinoid biosynthesis. This knowledge facilitates future targeted approaches towards Cannabis crop improvement and for the manipulation of cannabinoid metabolism.

Harnessing the advances of genetic engineering in microalgae for the production of cannabinoids.

Cannabis is widely recognized as a medicinal plant owing to bioactive cannabinoids. However, it is still considered a narcotic plant, making it hard to be accessed. Since the biosynthetic pathway of cannabinoids is disclosed, biotechnological methods can be employed to produce cannabinoids in heterologous systems. This would pave the way toward biosynthesizing any cannabinoid compound of interest, especially minor substances that are less produced by a plant but have a high medicinal value. In this context, microalgae have attracted increasing scientific interest given their unique potential for biopharmaceutical production. In the present review, the current knowledge on cannabinoid production in different hosts is summarized and the biotechnological potential of microalgae as an emerging platform for synthetic production is put in perspective. A critical survey of genetic requirements and various transformation approaches are also discussed.

Accumulation of somatic mutations leads to genetic mosaicism in cannabis.

Cannabis (Cannabis sativa L.) is typically propagated using stem cuttings taken from mother plants to produce genetically uniform propagules. However, producers anecdotally report that clonal lines deteriorate over time and eventually produce clones with less vigor and lower cannabinoid levels than the original mother plant. While the cause of this deterioration has not been investigated, one potential contributor is the accumulation of somatic mutations within the plant. To test this, we used deep sequencing of whole genomes (>50×) to compare the variability within an individual cannabis cultivar Honey Banana plant sampled at the bottom, middle, and top. We called over six million sequence variants based on a reference genome and found that the top had the most by a sizable amount. Comparing the variants among the samples uncovered that nearly 600,000 (34%) were unique to the top while the bottom only contained 148,000 (12%), and middle with 77,000 (9%) unique variants. Bioinformatics tools were used to identify mutations in critical cannabinoid-terpene biosynthesis pathways. While none were identified as high impact, four genes contained more than double the average level of nucleotide diversity (π) in or near the gene. Two genes code for essential enzymes required for the cannabinoid pathway while the other two are in the terpene pathways, demonstrating that mutations were accumulating within these pathways and could influence their function. Overall, a measurable number of intraplant genetic diversity was discovered that could impact long-term genetic fidelity of clonal lines and potentially contribute to the observed decline in vigor and cannabinoid content.

A Proteomic View of Cellular and Molecular Effects of Cannabis.

Cannabis (Cannabis sativa), popularly known as marijuana, is the most commonly used psychoactive substance and is considered illicit in most countries worldwide. However, a growing body of research has provided evidence of the therapeutic properties of chemical components of cannabis known as cannabinoids against several diseases including Alzheimer's disease (AD), multiple sclerosis (MS), Parkinson's disease, schizophrenia and glaucoma; these have prompted changes in medicinal cannabis legislation. The relaxation of legal restrictions and increased socio-cultural acceptance has led to its increase in both medicinal and recreational usage. Several biochemically active components of cannabis have a range of effects on the biological system. There is an urgent need for more research to better understand the molecular and biochemical effects of cannabis at a cellular level, to understand fully its implications as a pharmaceutical drug. Proteomics technology is an efficient tool to rigorously elucidate the mechanistic effects of cannabis on the human body in a cell and tissue-specific manner, drawing conclusions associated with its toxicity as well as therapeutic benefits, safety and efficacy profiles. This review provides a comprehensive overview of both in vitro and in vivo proteomic studies involving the cellular and molecular effects of cannabis and cannabis-derived compounds.

Characteristics of the Diploid, Triploid, and Tetraploid Versions of a Cannabigerol-Dominant F1 Hybrid Industrial Hemp Cultivar, Cannabis sativa 'Stem Cell CBG'.

Hemp (Cannabis sativa L.) has recently become an important crop due to the growing market demands for products containing cannabinoids. Unintended cross-pollination of C. sativa crops is one of the most important threats to cannabinoid production and has been shown to reduce cannabinoid yield. Ploidy manipulation has been used in other crops to improve agronomic traits and reduce fertility; however, little is known about the performance of C. sativa polyploids. In this study, colchicine was applied to two proprietary, inbred diploid C. sativa inbred lines, 'TS1-3' and 'P163', to produce the tetraploids 'TS1-3 (4x)' and 'P163 (4x)'. The diploid, triploid, and tetraploid F1 hybrids from 'TS1-3' × 'P163', 'TS1-3 (4x)' × 'P163', and 'TS1-3 (4x)' × 'P163 (4x)' were produced to test their fertilities, crossing compatibilities, and yields. The results indicated a reduction in fertility in the triploids and the tetraploids, relative to their diploid counterparts. When triploids were used as females, seed yields were less than 2% compared to when diploids were used as females; thus, triploids were determined to be female infertile. The triploids resulting from the crosses made herein displayed increases in biomass and inflorescence weight compared to the diploids created from the same parents in a field setting. Statistical increases in cannabinoid concentrations were not observed. Lastly, asymmetric crossing compatibility was observed between the diploids and the tetraploids of the genotypes tested. The results demonstrate the potential benefits of triploid C. sativa cultivars in commercial agriculture.

Origin and Evolution of the Cannabinoid Oxidocyclase Gene Family.

Cannabis is an ancient crop representing a rapidly increasing legal market, especially for medicinal purposes. Medicinal and psychoactive effects of Cannabis rely on specific terpenophenolic ligands named cannabinoids. Recent whole-genome sequencing efforts have uncovered variation in multiple genes encoding the final steps in cannabinoid biosynthesis. However, the origin, evolution, and phylogenetic relationships of these cannabinoid oxidocyclase genes remain unclear. To elucidate these aspects, we performed comparative genomic analyses of Cannabis, related genera within the Cannabaceae family, and selected outgroup species. Results show that cannabinoid oxidocyclase genes originated in the Cannabis lineage from within a larger gene expansion in the Cannabaceae family. Localization and divergence of oxidocyclase genes in the Cannabis genome revealed two main syntenic blocks, each comprising tandemly repeated cannabinoid oxidocyclase genes. By comparing these blocks with those in genomes from closely related species, we propose an evolutionary model for the origin, neofunctionalization, duplication, and diversification of cannabinoid oxidocycloase genes. Based on phylogenetic analyses, we propose a comprehensive classification of three main clades and seven subclades that are intended to aid unequivocal referencing and identification of cannabinoid oxidocyclase genes. Our data suggest that cannabinoid phenotype is primarily determined by the presence/absence of single-copy genes. Although wild populations of Cannabis are still unknown, increased sampling of landraces and wild/feral populations across its native geographic range is likely to uncover additional cannabinoid oxidocyclase sequence variants.

SNP in Potentially Defunct Tetrahydrocannabinolic Acid Synthase Is a Marker for Cannabigerolic Acid Dominance in Cannabis sativa L.

The regulation of cannabinoid synthesis in Cannabis sativa is of increasing research interest as restrictions around the globe loosen to allow the plant's legal cultivation. Of the major cannabinoids, the regulation of cannabigerolic acid (CBGA) production is the least understood. The purpose of this study was to elucidate the inheritance of CBGA dominance in C. sativa and describe a marker related to this chemotype. We produced two crossing populations, one between a CBGA dominant cultivar and a tetrahydrocannabinolic acid (THCA) dominant cultivar, and one between a CBGA dominant cultivar and a cannabidiolic acid (CBDA) cultivar. Chemical and genotyping analyses confirmed that CBGA dominance is inherited as a single recessive gene, potentially governed by a non-functioning allelic variant of the THCA synthase. The "null" THCAS synthase contains a single nucleotide polymorphism (SNP) that may render the synthase unable to convert CBGA to THCA leading to the accumulation of CBGA. This SNP can be reliably used as a molecular marker for CBGA dominance in the selection and breeding of C. sativa.

The genetics of Cannabis-genomic variations of key synthases and their effect on cannabinoid content.

Despite being a controversial crop, Cannabis sativa L. has a long history of cultivation throughout the world. Following recent legalization in Canada, Cannabis is emerging as an important plant for both medicinal and recreational purposes. Recent progress in genome sequencing of both cannabis and hemp varieties allow for systematic analysis of genes coding for enzymes involved in the cannabinoid biosynthesis pathway. Single-nucleotide polymorphisms in the coding regions of cannabinoid synthases play an important role in determining plant chemotype. Deep understanding of how these variants affect enzyme activity and accumulation of cannabinoids will allow breeding of novel cultivars with desirable cannabinoid profiles. Here we present a short overview of the major cannabinoid synthases and present the data on the analysis of their genetic variants and their effect on cannabinoid content using several in-house sequenced Cannabis cultivars.

Cannabis Research.

The International Cannabis Consortium (ICC) was founded in 2013 by Jacqueline Vink, Nathan Gillespie, Karin Verweij and Eske Derks. The largest contribution to the first meta-analysis was made by Prof. Nick Martin. The ICC has published two primary publications, in Translational Psychiatry and Nature Neuroscience, and many secondary publications. The study's principal investigators will always be grateful for Nick's contribution to science as they would not have been able to do any of this work without the contributions of Nick and others who collected samples. Nick has made unique contributions to the careers of many junior researchers by supporting their development and growth into senior positions.

The Interplay between the Endocannabinoid System, Epilepsy and Cannabinoids.

Epilepsy is a neurological disorder that affects approximately 50 million people worldwide. There is currently no definitive epilepsy cure. However, in recent years, medicinal cannabis has been successfully trialed as an effective treatment for managing epileptic symptoms, but whose mechanisms of action are largely unknown. Lately, there has been a focus on neuroinflammation as an important factor in the pathology of many epileptic disorders. In this literature review, we consider the links that have been identified between epilepsy, neuroinflammation, the endocannabinoid system (ECS), and how cannabinoids may be potent alternatives to more conventional pharmacological therapies. We review the research that demonstrates how the ECS can contribute to neuroinflammation, and could therefore be modulated by cannabinoids to potentially reduce the incidence and severity of seizures. In particular, the cannabinoid cannabidiol has been reported to have anti-convulsant and anti-inflammatory properties, and it shows promise for epilepsy treatment. There are a multitude of signaling pathways that involve endocannabinoids, eicosanoids, and associated receptors by which cannabinoids could potentially exert their therapeutic effects. Further research is needed to better characterize these pathways, and consequently improve the application and regulation of medicinal cannabis.

Cannabinoid Receptor Interacting Protein 1a (CRIP1a): Function and Structure.

Cannabinoid receptor interacting protein 1a (CRIP1a) is an important CB1 cannabinoid receptor-associated protein, first identified from a yeast two-hybrid screen to modulate CB1-mediated N-type Ca2+ currents. In this paper we review studies of CRIP1a function and structure based upon in vitro experiments and computational chemistry, which elucidate the specific mechanisms for the interaction of CRIP1a with CB1 receptors. N18TG2 neuronal cells overexpressing or silencing CRIP1a highlighted the ability of CRIP1 to regulate cyclic adenosine 3',5'monophosphate (cAMP) production and extracellular signal-regulated kinase (ERK1/2) phosphorylation. These studies indicated that CRIP1a attenuates the G protein signaling cascade through modulating which Gi/o subtypes interact with the CB1 receptor. CRIP1a also attenuates CB1 receptor internalization via ß-arrestin, suggesting that CRIP1a competes for ß-arrestin binding to the CB1 receptor. Predictions of CRIP1a secondary structure suggest that residues 34-110 are minimally necessary for association with key amino acids within the distal C-terminus of the CB1 receptor, as well as the mGlu8a metabotropic glutamate receptor. These interactions are disrupted through phosphorylation of serines and threonines in these regions. Through investigations of the function and structure of CRIP1a, new pharmacotherapies based upon the CRIP-CB1 receptor interaction can be designed to treat diseases such as epilepsy, motor dysfunctions and schizophrenia.

Disruption of an enhancer associated with addictive behaviour within the cannabinoid receptor-1 gene suggests a possible role in alcohol intake, cannabinoid response and anxiety-related behaviour.

The cannabinoid-1 receptor (CB1) plays a critical role in a number of biological processes including nutrient intake, addiction and anxiety-related behaviour. Numerous studies have shown that expression of the gene encoding CB1 (CNR1) is highly dynamic with changes in the tissue specific expression of CNR1 associated with brain homeostasis and disease progression. However, little is known of the mechanisms regulating this dynamic expression. To gain a better understanding of the genomic mechanisms modulating the expression of CNR1 in health and disease we characterised the role of a highly conserved regulatory sequence (ECR1) in CNR1 intron 2 that contained a polymorphism in linkage disequilibrium with disease associated SNPs. We used CRISPR/CAS9 technology to disrupt ECR1 within the mouse genome. Disruption of ECR1 significantly reduced CNR1 expression in the hippocampus but not in the hypothalamus. These mice also displayed an altered sex-specific anxiety-related behavioural profile (open field test), reduced ethanol intake and a reduced hypothermic response following CB1 agonism. However, no significant changes in feeding patterns were detected. These data suggest that, whilst not all of the expression of CNR1 is modulated by ECR1, this highly conserved enhancer is required for appropriate physiological responses to a number of stimuli. The combination of comparative genomics and CRISPR/CAS9 disruption used in our study to determine the functional effects of genetic and epigenetic changes on the activity of tissue-specific regulatory elements at the CNR1 locus represent an important first step in gaining a mechanistic understanding of cannabinoid regulatory pharmacogenetics.

A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci.

Cannabis sativa is widely cultivated for medicinal, food, industrial, and recreational use, but much remains unknown regarding its genetics, including the molecular determinants of cannabinoid content. Here, we describe a combined physical and genetic map derived from a cross between the drug-type strain Purple Kush and the hemp variety "Finola." The map reveals that cannabinoid biosynthesis genes are generally unlinked but that aromatic prenyltransferase (AP), which produces the substrate for THCA and CBDA synthases (THCAS and CBDAS), is tightly linked to a known marker for total cannabinoid content. We further identify the gene encoding CBCA synthase (CBCAS) and characterize its catalytic activity, providing insight into how cannabinoid diversity arises in cannabis. THCAS and CBDAS (which determine the drug vs. hemp chemotype) are contained within large (>250 kb) retrotransposon-rich regions that are highly nonhomologous between drug- and hemp-type alleles and are furthermore embedded within ∼40 Mb of minimally recombining repetitive DNA. The chromosome structures are similar to those in grains such as wheat, with recombination focused in gene-rich, repeat-depleted regions near chromosome ends. The physical and genetic map should facilitate further dissection of genetic and molecular mechanisms in this commercially and medically important plant.

Culture and cannabinoid receptor gene polymorphism interact to influence the perception of happiness.

Previous studies have shown that a cytosine (C) to thymine (T) single nucleotide polymorphism (SNP) of the human cannabinoid receptor 1 (CNR1) gene is associated with positive emotional processing. C allele carriers are more sensitive to positive emotional stimuli including happiness. The effects of several gene polymorphisms related to sensitivity to emotional stimuli, such as that in the serotonin transporter gene-linked polymorphic region (5HTTLPR), on emotional processing have been reported to differ among cultures-e.g., between those that are independent and interdependent. Thus, we postulated that the effects of the CNR1 genotype on happiness might differ among different cultures because the concept of happiness varies by culture. We recruited healthy male and female young adults in Japan, where favorable external circumstances determine the concept of happiness, and Canada, where the concept of happiness centers on positive inner feelings, and compared the effects of the CNR1 genotype on both subjective happiness levels (self-evaluation as being a happy person) and situation-specific happiness (happy feelings accompanying various positive events) by using a questionnaire. We found that the effect of CNR1 on subjective happiness was different between the Japanese and Canadian groups. The subjective happiness level was the highest in Japanese individuals with the CC genotype, whereas in Canadian participants, it was the highest in individuals with the TT genotype. Furthermore, the effects of CNR1 genotype on situation-specific happiness were also different between the groups. Happiness accompanied with being surrounded by happy people was the highest among Japanese individuals with the CC genotype, whereas among Canadian individuals, it was the highest in TT genotype carriers. These findings suggest that culture and CNR1 polymorphism interact to influence the perception of happiness.

Investigation of non-CB1, non-CB2 WIN55212-2-sensitive G-protein-coupled receptors in the brains of mammals, birds, and amphibians.

PURPOSE: Previous studies have found non-CB1 non-CB2 G-protein-coupled receptors in rodents that are activated by the aminoalkylindole cannabinoid agonist WIN55212-2. This work obtained evidence for the presence or absence of similar receptors in the brains of other mammals, birds and amphibians. MATERIALS AND METHODS: Antagonism of the stimulation of [35S]GTPγS binding by WIN55212-2 and CP55940 was assessed in multiple CNS regions of rat and canine, and in whole brain membranes from shrew, pigeon, frog and newt. A bioinformatics approach searched for orthologs of GRP3, GPR6, and GPR12 (closely related to cannabinoid receptors) in the genomes of these or related species. Orthologs were examined for amino acid motifs known to impart functionality to receptors. RESULTS: In mammals and pigeon, but not amphibians, a significant fraction of the stimulation of [35S]GTPγS binding by WIN55212-2 was not blocked by the CB1 antagonist SR141716A. BLAST searches found that GPR3 was restricted to mammals. GPR12 orthologs existed in all species, and they shared identical amino acid motifs. GPR6 orthologs existed all species, but with significant departures in the identity of some critical amino acids in bird, more so in amphibian. CONCLUSIONS: The portion of WIN55212-2-stimulated [35S]GTPγS binding that was antagonized by SR141716A was consistent with stimulation via CB1 receptors, indicating that antagonist-insensitive activity was via a different G-protein coupled receptor. Pharmacological evidence of this receptor was found in the brains of mammals and pigeon, but not frog or newt. Bioinfomatics results implicate GPR6 as a possible candidate for the additional WIN55212-2-sensitive receptor.

A new ESI-LC/MS approach for comprehensive metabolic profiling of phytocannabinoids in Cannabis.

Most clinical studies of Cannabis today focus on the contents of two phytocannabinoids: (-)-Δ9-trans-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD), regardless of the fact that the plant contains over 100 additional phytocannabinoids whose therapeutic effects and interplay have not yet been fully elucidated. This narrow view of a complex Cannabis plant is insufficient to comprehend the medicinal and pharmacological effects of the whole plant. In this study we suggest a new ESI-LC/MS/MS approach to identify phytocannabinoids from 10 different subclasses, and comprehensively profile the identified compounds in diverse medical Cannabis plants. Overall, 94 phytocannabinoids were identified and used for profiling 36 of the most commonly used Cannabis plants prescribed to patients in Israel. In order to demonstrate the importance of comprehensive phytocannabinoid analysis before and throughout medical Cannabis clinical trials, treatments, or experiments, we evaluated the anticonvulsant effects of several equally high-CBD Cannabis extracts (50% w/w). We found that despite the similarity in CBD contents, not all Cannabis extracts produced the same effects. This study's approach for phytocannabinoid profiling can enable researchers and physicians to analyze the effects of specific Cannabis compositions and is therefore critical when performing biological, medical and pharmacological-based research using Cannabis.