Abstract
In recent years, cannabidiol (CBD) has gained significant recognition in the medical field as a key component as an adjuvant in treatments for various conditions, including psychotherapeutic therapies. In Brazil, the cultivation and commercialization of Cannabis sativa, the plant from which CBD is derived, are subject to strict legal restrictions. This underscores the need to explore alternative methods of producing this compound without relying on the Cannabis plant. In response, we propose a project aimed at producing cannabidiol through synthetic biology. The compound will be synthesized using recombinant gene technology to express cannabidiolic acid (CBDA), the direct precursor of cannabidiol, in Saccharomyces cerevisiae. This process will require two metabolic pathways: the geranyl diphosphate (GPP) pathway, which is already present in yeast metabolism, and the olivetolic acid pathway. These precursors will be combined in two sequential enzymatic reactions to form cannabigerolic acid, which will then be converted into CBDA, which can be purified and converted into CBD. Ultimately, this proposal seeks to provide a more efficient method of producing CBD, free from the legal and bureaucratic challenges associated with Cannabis cultivation.
What is CBD?
CBD is one of the more than 100 phytocannabinoids present, originally, in Cannabis sativa. (Brunetti et al, 2020). It was first isolated from Cannabis in the late 1930s, and its structure was elucidated in 1964 by Gaoni and Mechoulam (Gaoni; Mechoulam, 1964). (Figure 1).
CBD is not a psychotropic agent, meaning it does not provoke any sensory alterations in the brain. This is possible since it does not activate the cannabinoid receptors CB1 and CB2 (Peng et al, 2022). Since the molecular mechanism of CBD is closely related to the endocannabinoid system present in humans (which plays a physiological role in regulating energy balance and processing fats and carbohydrates), its therapeutic action is of interest and widely studied (Mouslech, 2009; Pagotto et al, 2006). Figure 2 shows the signaling of the endocannabinoid system. This means it can be used as a therapeutic drug, once it has anti-inflammatory, sedative, antioxidant, anxiolytic, anticonvulsant and neuroprotective properties.
Medical Applications
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).
Conventional Way to Obtain Cannabidiol
The conventional way of obtaining cannabidiol is by extraction, purification and decarboxylation of CBD from plants of the genus Cannabis, which produce the compound naturally. CBD is extracted from the leaf and unfertilized flower of Cannabis sativa plants. The purification can be conducted in several different ways; this step is of vital importance since the extraction can remove other components other than cannabidiolic acid (CBDA), the direct precursor of CBD. Finally, thermal decarboxylation is employed, where heat breaks the bonds of CBDA’s acid group, removing it in the form of CO2 (Martinez et al, 2023).
In Cannabis sativa, cannabidiol is naturally synthesized through a process that utilizes substrates from two different metabolic pathways: the mevalonate pathway, which produces geranyl diphosphate (GPP), and the olivetolic acid pathway (Figure 3). 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).
Understanding the Problem
The problem with using CBD as a commercial drug thus lies in the social stigma and constitution of many countries around the plant, including Brazil. A portion of the country's population views the plant as harmful to social well-being, and Brazilian law treats Cannabis as an illicit substance and prohibits its recreational use. Even if under the same law (number 11.343, Brazilian Constitution of 1988) it’s staten that “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", what is seen in a day-to-day lifeset 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. There is a bill in the legislative chamber that aims to regulate the cultivation of Cannabis for medicinal purposes in the country, but it has been under discussion for over 5 years and has not yet been approved.
What has been done last year?
In order to bypass the extreme regulation over Cannabis sp. in the production of therapeutic drugs such as CBD, our proposal was to explore a synthetic alternative for its production using the yeast Saccharomyces cerevisiae. As stated, the production of CBD in Cannabis depends on two main pathways: the mevalonate and the olivetolic acid. Our choice on S. cerevisiae lies on the fact that it already has, naturally, the mevalonate pathway in its metabolism. Consequently, the goal was to express the olivetolic acid pathway in our yeast, enabling it to produce the cannabidiolic acid (CBDA) - the direct precursor of CBD. This would offer a promising solution to favor the access to CBD in Brazil.
The initial focus in 2023 was on project development using mathematical modeling tools, enabling us to consolidate the proof of concept and devise wet-lab strategies for constructing the necessary biological circuits (See 2023 Wiki). From this foundation, we developed genetic circuits to introduce the Cannabis biosynthetic pathway into Saccharomyces cerevisiae. Structural modeling of the enzymes, phylogenetic analyses, molecular docking, and expression evaluations were conducted to guide the development and optimization of these biological circuits. Moreover, the choice of S. cerevisiae as the chassis was strategic, as it naturally synthesizes geranyl diphosphate (GPP), requiring only the introduction of the olivetolic acid pathway for CBDA production. Additionally, S. cerevisiae is widely used in industrial applications. To ensure the efficient synthesis of the five enzymes involved in the biological circuit, we employed T2A sequences, which allow for the production of multiple proteins from a single mRNA molecule. This method takes advantage of the inability of the eukaryotic ribosome to form peptide bonds between the terminal residues of proline and glycine, thereby generating separate proteins without interrupting protein synthesis (Mansouri, 2014).
NEW CHALLENGES
This year, we're focused on the wet-lab part proposed in the last year and on how we can improve and optimize this production to make it competitive with plant-based production. To achieve this, we re-analyzed the mevalonate pathway, which produces the precursor for the synthetic pathway: geranyl-PP (GPP). With this in mind, we developed methods to accumulate GPP in the cell and ensure it is entirely converted to CBDA, which can then be subsequently converted to CBD.
For this optimization, the team designed an RNA interference cassette and an overexpression cassette so that, in addition to producing CBDA in yeast, we could also enhance this production using other molecular and synthetic biology techniques. In these cassettes, we used two main enzymes: ERG20, responsible for converting GPP to farnesyl-PP (FPP), and GPPS, responsible for generating GPP. By decreasing the expression of ERG20 and increasing the expression of GPPS, we can accumulate our main metabolite, and consequently increase the production of CBDA by the yeast.
Additionally, to complement the modeling work done last year, this year we developed kinetic modeling for the five main enzymes involved, evaluating parameters such as reaction rate in relation to the added substrate. This allows us to obtain better analysis of the structures we are working with and, consequently, achieve better results in the wet lab.
Furthermore, to scale up our project's production, we are developing a low-cost bioreactor so that, along with the other improvements made, the yeast can produce a satisfactory amount of CBDA at a larger scale and with low production costs, making our product more competitive and applicable to real life.
OUR GOALS
- Construct overexpression, deletion, and/or gene silencing cassettes in S. cerevisiae with a goal to optimize the production of CBD.
- Obtain S. cerevisiae strains transformed with the plasmids of interest.
- Perform gene expression assays with the transformed and mutant strains.
- Kinetic modeling of the five enzymes studied last year, to ensure better analysis of the structures.
- Develop a low-cost bioreactor that would allow the production of a decent amount of CBDA by the mutant S. cerevisiae strains.
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