They demonstrated that ∆9 -THC synthetic mimics were able to bind to a specific site in the membranes of the brain, inhibiting synthesis of cyclic adenosine monophosphate through a G-protein mediated mechanism. Furthermore, molecular cloning of the first cannabinoid receptor gene as well as brain mapping of the cannabinoid binding sites in rats confirmed the existence of the cannabinoid receptor in the brain, known as the CB1 receptor. The cannabinoid receptors are part of a larger family of receptors known as G-protein coupled receptors . G-protein coupled receptors total about 800 in number and are divided into five major families: secretin, frizzled/taste 2, adhesion, glutamate, and rhodopsin. Majority of GCPRs including CB1 and CB2 belong to the rhodopsin like family. Rhodopsin is a pigment found in the rod photoreceptor cells of the retina and its role is to change photons into chemical signals, allowing vertebrate animals and humans to sense light by stimulating cellular biological processes in these organism’s central nervous systems. With regards to structure, GCPRs contain a transmembrane unit harboring seven alpha-helices conjoined with a G-protein that itself has three subunit proteins: Gα, β, and γ. Once an agonist binds to the GCPR transmembrane domain, the G-protein subunits conjoin with another cellular protein such as a protein kinase or adenyl cyclase to catalyze downstream functions. GCPRs role in regulating functions in the cells is so important that drugs targeting GCPRs make up 30- 40% of all the drugs in the market. 20 The CB1 receptor’s discovery was in 1984 through the observation that cannabinoid binding to the receptors reduced cyclic adenine monophosphate concentrations in neuroblastoma cells.
Two years later,seedling starter trays work was done to demonstrate that the cAMP concentration reduction by cannabinoid binding could be reversed through the exposing of the cells to the pertussis toxin, a Gαi protein inhibitor. However, it wasn’t until 1990 that the CB1 receptor was cloned and elucidated from a cDNA library of cerebral cortex tissues with studies that same year demonstrating that CB1 receptors in the brain were nearly as prevalent as gammaaminobutyric acid receptors and glutamate receptors. The prevalence of CB1 receptors and their localization in the brain allowed researchers to correlate their expression to the subsequent pharmacological effects. For example, localization and expression of CB1 in the hippocampus and cerebral cortex correlated to memory and cognition effects whereas localization and expression of CB1 in the cerebellum and basal ganglia was correlated to stride or gait effects. Therefore, binding of the CB1 receptors in the brain correlates to the psychoactive effects of cannabis. Although CB1 receptors are prevalent in the brain, they also have been found to be located in the uterus, prostrate, adrenal glands, tonsils, gastrointestinal tract, spleen, and vascular smooth muscle cells. The discovery of the CB2 receptor answered the question of why cannabis was reported to have immunomodulatory effects. The CB2 receptor was discovered three years after the CB1 receptor and was found in a human promyelocytic leukemia cell line. Unlike the well characterized CB1 receptor, CB2 has not been as well as characterized due to numerous conflicting reports about the effects of its expression but one thing is known for sure: the CB2 receptor plays a strong role in immunomodulatory effects with great implication for example, in Alzheimer’s and Huntington’s disease. The CB2 receptor is known as the peripheral receptor for cannabinoids and is primarily found and expressed in immune tissues.
When these cannabinoid receptors are activated, as previously mentioned with regards to CB1, there is a decrease in cAMP levels and there is also modulation of potassium and calcium levels in the cells. When these receptors are stimulated, p38 mitogen activated protein kinases , c-Jun N-terminal kinases, and p42 and p44 MAPKs are activated. The p42/44 MAPKS are also referred to as extracellular signal regulated kinases 1 and 2 and they are involved in transcription regulation, cell differentiation, downstream regulation of genes, and cytokine synthesis regulation.Both cannabinoid receptors utilize the transducing G proteins, G1 and Go, responsible for a wide range of cellular functions such as response to environmental stimuli and responses to hormonal signals. ∆ 9 -THC binding to these receptors causes the opening of potassium channels, inhibition of adenylyl cyclase activity, closing of voltage gated calcium channels, and stimulation of mitogen-activated protein kinases, among other responses. The characterization of these cannabinoid receptors in human signaled to researchers that there are likely some endogenous ligands capable of binding to these receptors. These ligands are part of the endocannabinoid system and include anandamide which was found to produce similar effects as the cannabinoids from Cannabis sativa, O-arachidonoyl ethanolamine, and 2- arachidonoylglycerol , and 2-arachidonoyl glycerol ether. Additionally, reports have been made indicating that cannabinoids have been able to bind to other receptors in the body outside of CB1 and CB2. Cannabinoids have been found to be capable of binding to the transient receptor potential cation channel vanilloid type 1 , 5- hydroxytryptamine -3A ligand-gated ion channel, G-protein-coupled receptor 55 , 5-hydroxytryptamine -3A ligand-gated ion channel, and transient receptor potential cation channel Ankyrin type 1 . Both ∆ 9 -THC and CBD were isolated from the hemp oil plant in the 1940s. Unlike ∆ 9 -THC, CBD has no psychoactive properties and has low affinity to both CB1 and CB2 receptors, whereas ∆9 -THC binds effectively to the CB1 receptor.
Although CBD has low binding affinity to the endocannabinoid receptors, there is some data that indicates that CBD has some beneficial properties in the treatment of seizures and epilepsy, movement disorder, psychosis, anxiety, multiple sclerosis, and Huntington’s disease, once again highlighting the therapeutic potential of cannabinoids. The therapeutic potential of cannabinoids has led to the development of cannabinoidbased medicines . Currently, in the United States, there are 3 licensed CBMs approved by the Food and Drug Administration: nabilone , a synthetic analog of ∆9 -THC, used as an antiemetic, preventing vomiting and nausea caused by cancer medications, dronabinol, synthetic ∆9 -THC , used to treat lack of appetite leading to weight loss in AIDS victims as well as treat nausea and vomiting like nabilone, and a liquid formulation of dronabinol. The FDA has also placed on the fast track a few more CBMs and has also approved investigational drug studies of CBD due to its ability to treat chronic pain and help prevent seizures in childhood epilepsy cases. All in all, the global pharma cannabinoid industry is projected to exceed $102 billion by 2030 indicating a significant interest in the development of CBMs; therefore, methods for the efficient large-scale production of cannabinoids are necessary.In recent years, there has been significant interest from the synthetic biology community to produce cannabinoids using microbial and cell-free strategies because of the flexibilities in engineering the pathway to access rare or unnatural cannabinoids, the challenges associated with chemical synthesis, and the inconsistent and relatively low production of cannabinoids from plants.Owing to the identification of important enzymes in the cannabinoid biosynthesis pathway such as olivetolic acid synthase and olivetolic acid cyclase , which converts hexanoyl-CoA to olivetolic acid, microbial hosts have been utilized to produce cannabinoids.Another key finding was the discovery of the prenyltransferase responsible for prenylating olivetolic acid with geranyl to form cannabigerolic acid . With these key enzymes identified, botanicare trays different groups have attempted to microbial produce cannabinoids and their precursors. Tan et al., employing olivetolic acid synthase and olivetolic acid cyclase from the Cannabis sativa plant in addition to further engineering, were able to produce 80 mg/L of olivetolic acid from E. coli. To date, that is the highest literature recordedamount of olivetolic acid produced in vivo in any wild type or engineered organism. Additionally, Luo et al. were able to express the entire cannabinoid pathway in Saccharomyces cerevisiae and produce 8 mg/L of tetrahydrocannabinolic acid , the direct ∆9 -THC precursor, and 4.3 ug/L of cannabidiolic acid, the direct CBD precursor; this was the first time that ∆9 -THC and CBD were produced by yeast. Furthermore, Valliere et al., utilizing commercial olivetolic acid and the synthetic biochemistry approach of cell-free systems, showed that they were able to produce 1.25 g/L of cannabigerolic acid , by engineering an aromatic prenyltransferase from Streptomyces sp. CL190 to efficiently prenylate olivetolic acid, another important intermediate in the cannabinoid biosynthesis platform. 42 Additionally, Jimbo Ma et al engineered the yeast Yarrowia lipolytica to improve the biosynthesis of olivetolic acid and achieved an 83 fold titer increase giving a final titer of .11 mg/L. 43 E.coli and yeast strains have not been the only microbial organisms utilized to produce olivetolic acid and cannabinoids. Two groups have demonstrated that amoeba can also be engineered for the production of olivetolic acid. Reimer et al. engineered Dictyostelium discoideum, due to its ability to produce polyketides and terpenoids, to ultimately produce olivetolic acid. By fusing the OLS to the C-terminal of the D. discoideum StII gene , they were able to engineer an amoeba/plant hybrid gene for the production of olivetolic acid. The group utilized the StII gene, an enzyme consisting of a type III PKS and FAS and swapped the type III PKS domain with OLS and overexpressed this fused hybrid gene to produce olivetolic acid.
Kufs et al also utilized Dictyostelium discoideum to produce olivetolic acid , developing a scaled approach in bioreactors able to achieve a titer of 4.8 µg/L. Although novel with the use of Dictoystelium discoideum to produce this cannabinoid intermediate, such a result underscores the need to increase production of olivetolic acid. To date, these approaches for the production of olivetolic acid and cannabinoids all rely on the plant pathway with the enzymes olivetolic acid synthase and olivetolic acid cyclase catalyzing the formation of olivetolic acid from hexanoic acid and have heavy intellectual property surrounding them so a novel way of producing olivetolic acid is preferred. We were able to discover a novel pathway to olivetolic acid by focusing on fungal natural product bio-synthetic pathways, a vast field with promising capabilities. Natural products from fungi have demonstrated great therapeutic and agricultural potential with two of the currently used anti-fungal drugs being the natural products echinocandins and amphotericin. Echinocandins, referred to as the “penicillin of antifungals”, and amphotericin are also included in the World Health Organization’s List of Essential Medicines, showcasing the powerful potential of fungal natural products. Natural products, also referred to as secondary metabolites, are most times not essential for the organism’s life but do still have important roles such as acting as metal-transporting agents, symbiosis facilitators, sexual and differentiation effectors, and metal-transporting agents, among other functions. Fungi produce a vast variety of natural products that can be classified as terpenes, polyketides, sugars, and alkaloids. There are 100,000 known fungal species, although upwards of one million fungal species are expected to be in existence. Of the known 100,000 fungal species, only a fraction of their natural products and bio-synthetic pathways have been elucidated. Therefore, there is great potential to search for more fungal natural products with interesting bio-activities, underscoring the importance of genome mining. These bio-synthetic pathways encode secondary metabolite genes that produce natural compounds. Genome mining has been used to search for bio-synthetic pathways for known products as well as undiscovered bio-synthetic pathways that can produce novel natural products. Genome mining answers the question of how natural products are formed. It describes the utilization of genomic information to search for bio-synthetic gene clusters responsible for producing natural products. Over the years many different methods of genome mining have been developed. Among them include classical genome mining, comparative genome mining, phylogeny-based genome mining, and resistance gene/target directed genome mining. Classical genome mining involves the search for genes involved in a bio-synthetic pathway. Typically, the process consists of querying for a desired gene across many genomes and then querying for that gene in the context of a bio-synthetic gene cluster. NCBI Basic Local Alignment Search Tool is widely utilized for classical genome mining. Comparative genome mining involves comparing multiple genomes in different organisms to identify similar clusters. It differs from classical genome mining in that instead of searching for single genes, one is searching for partial or whole gene clusters across various genomes. Gene clusters in a genome are then prioritized based on their homology to other clusters in other organisms’ genomes.