In recent years, cannabidiol has gained ground in medicine as a component of medicaments for countless treatments, such as psychotherapeutic and oncological treatments. In Brazil, Cannabis sativa, the plant from which CBD is extracted, has several legal restrictions on its cultivation and commercialization. It becomes evident, therefore, the necessity to diversify ways of obtaining this product without the need for the Cannabis plant. Thus, we propose this project, which has as its main objective the production of cannabidiol via synthetic biology. The compound will be obtained using recombinant gene technology to express the direct precursor of cannabidiol, cannabidiolic acid (CBDA) using Saccharomyces cerevisiae. Two metabolic pathways will be necessary for this: the geranyl diphosphate (GPP) pathway, already present in the yeast metabolism, and the olivetolic acid pathway. Both precursors will be combined in two subsequent enzymatic reactions steps to form cannabigerolic acid, which will subsequently be converted into cannabidiolic acid, which can be purified and converted into CBD. Finally, the proposal for the production of CBD without using Cannabis plants is expected to provide a less bureaucratic and expensive method of obtaining the drug.
Cannabidiol (CBD) is a phytocannabinoid that constitutes 40% of the Cannabis sativa extract. CBD is a non-psychotropic agent, i.e., it exhibits pharmacological actions without causing sensory alterations in the brain or compromising other internal functionalities, since it does not activate the cannabinoid receptors CB1 and CB2 (Sousa; Baião , 2021). In 1964, using chromatography, the structure of the compound was solved and published for the first time (Gaoni; Mechoulam, 1964).
Figure 1: First structure published of Cannabidiol.
Source: 1964, Gaoni and Mechoulam
Cannabidiol should be kept without contact of oxygen and light, since it is susceptible to oxidation and photochemical reactions (Mechoulam and Hanus, 2002). The oxidation reaction of CBD occurs in a basic solution with the presence of oxygen, forming monomeric and dimeric hydroxy-quinones (Mechoulam et al., 1968). Photochemical reactions are varied, including the formation of isomeric 1-methoxy dihydro CBD when cannabidiol is present in an organic solution, as the case for methanol (Shani and Mechoulam, 1971).
The conventional way to obtain cannabidiol is by extraction, purification and decarboxylation of CBD from plants of the genus Cannabis, which produce the compound naturally. In Cannabis sativa, naturally, cannabidiol is produced in a pathway that uses as substrates the products of two other metabolic pathways: the mevalonate pathway, which generates geranyl diphosphate (GPP), and the olivetolic acid pathway (Zirpel et al., 2017).
Through the olivetolic acid pathway, CBD synthesis happens in five steps. The first step is the activation of hexanoic acid by Hexanoyl-CoA synthase. The second step is the generation of olivetol, in a reaction catalyzed by the enzyme olivetol synthase. The third step is the conversion of olivetol to olivetolic acid with action of the enzyme olivetolic acid synthase. The fourth step is the prenylation of the olivetolic acid produced in the previous step, in a reaction catalyzed by the enzyme cannabigerolic acid synthase. The fifth and final step is the conversion of cannabigerolic acid to CBDA in a reaction catalyzed by CBDA synthase (Zirpel et al., 2017).
Figure 2: Synthesis reaction of cannabigerolic acid, catalyzed by a prenyltransferase.
Source: 2023, KEGG Pathway Database adapted.
The first successful research into the isolation of CBD dates back to 1940. From an ammonolysis of bis-Z, 5-dinitrobenzoate purified by means of ammonia in toluene with cannabis extract, a pure component was isolated and named cannabidiol (Adams; Hunt; Clark, 1940).
Currently, CBD is extracted from the leaf and unfertilized flower of plants of the Cannabis genus (Vargas, 2021). The compound can be extracted and purified in several different ways, which are seen in the chart below (Figure 3). Among the various methods, the most common are those that use solvents, and in this section the most commonly used is supercritical carbon dioxide gas (scCO2). Under conditions of very high pressure, carbon dioxide becomes liquid at low temperatures and consequently the compound can be used as a solvent (Martinez et al., 2023).
Figure 3: Reported techniques for the extraction of valuable compounds from various parts of the Cannabis plant.
Source: 2023, Martinez et al..
After extraction of total compounds from Cannabis, purification of the obtained product is necessary, since this method extracts components other than cannabidiolic acid (CBDA), the acid form and precursor of CBD. Therefore, the processes of CBDA separation and decarboxylation are important. The fulfillment of these objectives is accomplished through separation processes, usually filtration and distillation, in order to isolate the desired compound, and the operation of thermal decarboxylation, where heat promotes the breaking of bonds of the acid group, that is, the compound in question is heated and, consequently, the acid group is removed from the molecule in the form of CO2. (Martinez et al, 2023)
Cannabidiol (CBD) is a phytocannabinoid with little to no psychoactive effect and abundant in plant extracts of the Cannabis genus, possessing a high affinity for human receptors such as cannabinoid types 1 (CB1) and 2 (CB2) (Peng et al., 2022). As the molecular mechanism of CBD is closely related to the human endocannabinoid system (an internal communication system that plays a physiological role in regulating energy balance and processing fats and carbohydrates), the study of its therapeutic action for the treatment of pathologies brings new perspectives to the scientific community (Mouslech, 2009; Pagotto et al., 2006). Figure 4 shows the signaling of the endocannabinoid system.
Figure 4: Signaling of the endocannabinoid system
Source: 2016, Hiley & Ford (2004) apud Flores
Presenting several beneficial effects, as it has anti-inflammatory, sedative, antioxidant, anxiolytic, anticonvulsant, and neuroprotective properties, the use of CBD is of great medical interest for the therapeutic treatment of diseases ranging from inflammatory to neurodegenerative (Flores, 2016; Donk, 2019).
Therefore, its importance is known in the treatment of diseases, especially chronic and psychiatric, with the use of cannabidiol (CBD). Among them, the use of CBD is more recurrent in the treatment of anxiety disorders, depression, schizophrenia, multiple sclerosis and, in a more widespread and incisive way, epilepsy (Alves et al., 2020).
Epilepsy represents a significant therapeutic challenge, because, even with the increase in the number of anticonvulsant drugs available, about one third of patients continue to face persistent seizures. Hence, this reality highlights the existence of a gap in the current treatment, which encourages advances in the research of new anticonvulsant drugs, such as cannabidiol (CBD), in the search for alternatives that can provide better seizure control and offer more therapeutic options to affected patients (Silva et al., 2020).
The effectiveness of CBD starts from the agonist action towards TRPV1 channels, resulting in the desensitization of these channels and then normalization of intracellular calcium levels. In addition, CBD interacts with other T-type calcium ion channels, which play a role in regulating neuronal excitability. In conditions such as epilepsy there is activation of these channels due to a hyperpolarization of neuronal membranes, which leads to an increase in intracellular calcium concentration, increasing the excitability of neurons. However, CBD blocks the function of these T-type calcium channels, suggesting that this mechanism is responsible for its antiepileptic action (Silvestro et al., 2019).
In Brazil, the social stigma related to Cannabis sativa, the raw material of cannabidiol, is cultural. A portion of the country's population views the plant as a harmful drug to social well-being. This prejudice is reflected in the law, which treats Cannabis as an illicit substance and prohibits its recreational use. However, in the same law, number 11.343, it states, "The Union may authorize the planting, cultivation, and harvesting of the plants referred to in the caput of this article, exclusively for medicinal or scientific purposes, in a predetermined location and timeframe, subject to supervision, respecting the aforementioned reservations" [Brazilian Federal Constitution (1988)]. In other words, there are significant bureaucratic obstacles to the use of Cannabis sativa substrate for medicinal purposes. CBD is subject to various taxes, contributing to an increase in the price of its products. However, there is a bill in the legislative chamber that aims to regulate the cultivation of Cannabis for medicinal purposes in the country, but this bill has been under discussion for over 5 years and has not yet been approved.
Through the development of the project, we expect to:
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