This study significantly contributes to the body of research on cannabidiol (CBD) and provides valuable insights for future researchers by consolidating findings across various topics related to CBD. By comparing the bioavailability of CBD in both standalone and liposomal formulations, the study emphasizes the importance of formulation in enhancing absorption and effectiveness. Such insights can guide subsequent research efforts in exploring optimal delivery systems for CBD, paving the way for more effective therapeutic applications.
Moreover, this research highlights critical challenges associated with CBD’s physicochemical properties, such as low water solubility and sensitivity to environmental factors. Addressing these challenges will be crucial for future studies focused on developing innovative extraction, purification, and delivery methods. As researchers build upon this foundation, they can explore the implications of CBD’s antibacterial properties, potential applications in various medical fields, and the importance of sustainable production practices.
Overall, by consolidating knowledge from multiple studies and providing a clear pathway for further investigation, this research serves as a valuable resource for future scientists and practitioners in the field of cannabinoid research and biotechnology.
The iGEM USP-EEL-Brazil 2024 team is excited to share the results and findings from our two-year project focused on optimizing cannabidiol (CBD) production. A crucial part of our work involved the kinetic modeling of key enzymes in the CBD biosynthesis pathway. By providing detailed enzyme velocity profiles, we aim to offer a valuable contribution to future iGEM teams and facilitate the replication and adaptation of our methodologies.
We determined the velocity profiles for several essential enzymes in the process. Cannabidiolic Acid Synthase (CBDAS), a critical enzyme from Cannabis sativa of the fiber type, catalyzes the oxidative cyclization of cannabigerolic acid (CBGA) to form cannabidiolic acid (CBDA), the precursor to CBD. CBDAS was characterized as a covalently flavinylated oxidase and expressed in insect cell cultures, showing catalytic activity. This enzyme requires molecular oxygen and produces hydrogen peroxide during the reaction, similar to tetrahydrocannabinolic acid synthase (THCAS). The values obtained for CBDAS were a Km of 0.137 mM and a Vmax of 2.57 nmol/sec/mg.
Hexanol-CoA Synthase (CsHCS1), identified as CsAAE1, plays a fundamental role in cannabinoid biosynthesis by synthesizing Hexanol-CoA, an essential precursor. Located in the cytoplasm of glandular trichome cells, CsHCS1 primarily activates short-chain fatty acids such as hexanoate. We obtained a Km of 3.7 µM and a Vmax of 6.8 pKat/g for this enzyme. CsHCS1 does not follow the Michaelis-Menten profile due to substrate inhibition, and therefore, we applied a nonlinear regression with substrate inhibition for these values.
Olivetolic Acid Geranyltransferase/Prenyltransferase 4 (CsaPT4/PT4) demonstrated geranyltransferase activity and was capable of producing cannabigerolic acid (CBGA) from olivetolic acid and geranyl pyrophosphate (GPP). Functionally expressed in yeast, CsPT4 has a Km of 5.7 μM and a Vmax of 0.1 μmol/g/min, revealing its efficiency in CBGA production.
For Olivetol Synthase/3,5,7-Trioxododecanoil-CoA Synthase (OS/OLS), a type III polyketide synthase enzyme, we found a Km of 60.8 μM. OLS synthesizes olivetol from hexanoil-CoA and malonyl-CoA and is expressed in rapidly expanding flowers and leaves of Cannabis sativa. OLS accepts CoA esters with aliphatic side chains of C4 to C8, with a preference for hexanoil-CoA. We determined a Vmax of 5.792 nmol/sec for OLS.
Regarding Olivetolic Acid Cyclase (OAC), we chose not to calculate Km and Vmax values due to its function as a DABB family enzyme, which performs a direct substrate conversion. In our kinetic model, OAC was considered as performing an immediate conversion, simplifying the analysis by focusing on OLS as the main bottleneck.
We are making all related data and graphs available to support other teams in applying these techniques to their projects. We believe this information will be extremely useful to the iGEM community, advancing the optimization of biotechnological processes and contributing to the progress of biosynthesis projects.
We remain available to collaborate and share knowledge with the iGEM community and hope that our findings inspire and assist other teams in achieving their goals.
| Enzyme | Mol. wt (kDa) | Protein Family | Catalytic reaction | Substrate | Kinetic parameters |
|---|---|---|---|---|---|
| CBDAS | 62.237 | oxygen-dependent FAD-linked oxidoreductase family | stereoselective oxidative cyclisation of CBGA | CBGA | KM =0.137 mM, Vmax = 2.57 nmol/s/mg |
| CsHCS1 | 79.715 | AAE (Acyl-Activating Enzyme), a class of acyl-CoA synthetases | Activation of hexanoate to form Hexanoyl-CoA | Hexanoate (hexanoic acid) | Km = 3.7 mM, Vmax = 6.8 pKat |
| PT4 | 44.928 | Transferase family (geranyltransferases) | Transfer of geranyl to olivetolic acid, forming cannabigerolic acid | Olivetolic acid and geranyl pyrophosphate (GPP) | Km = 5.7 μM, Vmax = 0.1 μmol/g/min |
| Olivetol synthase (OLS) | 42.585 | Thiolase-like superfamily (Chalcone/Stilbene synthases) | Aldol condensation; Water activation by hydrogen bonding network | Hexanoyl-CoA | Km = 60.8 μM, kcat = 2.96 min-1 |
| Olivetolic acid cyclase (OAC) | 12.002 | dimeric α+β barrel (DABB) | Acid-base catalytic chemistry, C2–C7 aldol cyclisation | 3,5,7-trioxododecanoyl-CoA | not found |
To streamline the calculation process and ensure the accuracy of our project, we developed a highly adaptable Python© code capable of performing all the necessary calculations automatically. This was a critical step in optimizing our workflow and reducing the likelihood of human error, especially considering the complexity and variety of calculations required for different aspects of our work.
A key feature of the code is its user-friendly nature. We recognized that not everyone in our team, or those who may want to use the code in the future, would have a background in programming. Therefore, we paid special attention to ensuring that the code is well-documented and easy to navigate. By adding a large number of detailed comments throughout the code, we provided clear explanations of each step, function, and calculation. This approach was designed so that even users with little or no programming knowledge could understand what each part of the code does.
Furthermore, users can easily make adjustments to suit their specific needs, such as changing parameters, altering concentration values, or modifying the conversion of units. Although all our calculations were carried out using the Metric System, the code allows for straightforward unit conversions if necessary. This flexibility ensures that the code can be used in a wide range of contexts and adapted to different scientific or educational purposes.
We believe that by sharing this tool, we are contributing to a broader culture of open science and collaborative learning, empowering others to use, modify, and learn from our approach. You can check it out at our wiki's repository under "Bioreactor Codes"
The iGEM allows teams to work together in a collaborative space, where they can share data and use results from previous research and projects. As members of the USP-EEL-Brazil team, we are excited and honored to be able to help and provide new resources and information to future competitions, contributing to the dissemination of knowledge and the promotion of a supportive and united environment. This year, the standout collaboration is the synthetic pathway of phytocannabinoids. Since the CBD pathway is similar to other pathways of compounds present in Cannabis, components that are also very effective medicinally, such as THC, for example, could also be produced by simply swapping the final enzyme in the circuit developed in the project. Therefore, this material could be useful for teams that work in the future with derivatives of Cannabis sativa, as well as our research and analyses on the intense methodologies and bureaucracies related to this plant in numerous countries.
Furthermore, the results of the data collection on female presence in iGEM teams may be useful for future generations; just as we were inspired by the Paris Bettencourt team, other teams may be inspired by our research, possibly analyzing what has happened over the years in this regard.
Moreover, we believe that the method by which we promote inclusion in science, developing fun and engaging materials adapted to each context, is inspiring. We aim not only to reach different age groups but also people from various social backgrounds and with diverse realities. Our materials are available not only to the Brazilian population but also to other iGEM teams that seek to make a difference
This book is aimed at children and was distributed as part of the ImagineNation project. Its purpose is to educate preschool-aged children about various microorganisms and their functions, while also promoting the importance of handwashing in a playful and fun way. This is achieved through the introduction of characters representing microorganisms, as well as our own mascots, named "Lelê" and "Durinha," which reference the term “levedurinha” (little yeast in Portuguese).
This book was developed with an older audience in mind, targeting pre-teens and teenagers, for distribution and promotion at the Cotel fair. The concept behind this material is the presentation of fundamental concepts for understanding synthetic biology, organized in a standardized format according to the letters of the alphabet. We managed to introduce at least one concept for each letter, as well as renowned names in the field, always aiming to reflect our inclusive purpose by highlighting scientists from minority groups in the synthesized content.
This game targeted children’s interest in fundamental principles of synthetic biology, such as promoters, coding sequences, ribosome binding sites and terminators. The simple concept mixed with colorful and engaging designs managed to capture the attention of our public while also helping them learn and flourish their creativity.
We’ll make our information cards, used to explain the theory behind the game, and the pieces needed to play it available for download. This way, iGEM teams and anyone interested in getting inspired by or playing our games can easily find them.
This game was inspired by the original mafia game, which involves citizens trying to expose assassins that are hidden among the crowd. However, in order to correlate this dynamic with synthetic biology, the assassins use a virus instead and, in each round, players get contaminated, scientists try to develop a cure and citizens try to find out who the assassins are.
Information cards regarding this activity will also be available, meaning to help new people to understand how the various types of players can act and reach even more individuals with fun science-based games.
By the time of the publication of this website, our team developed four unique adapters to allow the lab to become a more inclusive and diverse space.
This tool is targeted towards people with disabilities or low physical mobility. It has three sizes of beakers in mind (small, medium and large), aiming to provide stabilizing support for this type of apparatus, though a circle platform in which the beaker should be placed upon, rather than being placed on a slippery surface that can be troublesome and unwelcoming to many potential scientists.
This adapter is also targeted to help people with disabilities and low physical mobility to manipulate lab utensils more easily and efficiently. The support, when attached to the tip of the glass stick, increases the holding diameter and allows a better grip, since the texture and material are more comfortable to the touch compared to the usual glass used in this type of device,
This device was designed as a board that has an embossed part on it, meant to show, in a tactile manner, whether the UV light is turned on or not. The easiest way to add texture to this board is through the use of velcro. When turned on, the person responsible for the process should attach the velcro to the embossed part and, once it’s done, remove it and put it on the recessed area of the board. This way, people will be able to safely communicate in the lab and make sure everyone is being taken into consideration, for the sake of inclusivity and safety.
This initiative has the goal to make individuals with low vision more independent, safe and aware of the risks and precautions associated with both the chemical components that are more common and dangerous.
Taura, F., Sirikantaramas, S., Shoyama, Y., Yoshikai, K., Shoyama, Y., & Morimoto, S. (2007). Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-typeCannabis sativa. FEBS Letters, 581(16), 2929–2934. https://doi.org/10.1016/j.febslet.2007.05.043
Gagne, S. J., Stout, J. M., Liu, E., Boubakir, Z., Clark, S. M., & Page, J. E. (2012). Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proceedings of the National Academy of Sciences, 109(31), 12811–12816. https://doi.org/10.1073/pnas.1200330109
Luo, X., Reiter, M. A., d’Espaux, L., Wong, J., Denby, C. M., Lechner, A., Zhang, Y., Grzybowski, A. T., Harth, S., Lin, W., Lee, H., Yu, C., Shin, J., Deng, K., Benites, V. T., Wang, G., Baidoo, E. E. K., Chen, Y., Dev, I., & Petzold, C. J. (2019). Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature, 567(7746), 123–126. https://doi.org/10.1038/s41586-019-0978-9
Taura, F., Tanaka, S., Taguchi, C., Fukamizu, T., Tanaka, H., Shoyama, Y., & Morimoto, S. (2009). Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway. FEBS Letters, 583(12), 2061–2066. https://doi.org/10.1016/j.febslet.2009.05.024
Stout, J. M., Boubakir, Z., Ambrose, S. J., Purves, R. W., & Page, J. E. (2012). The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes. The Plant Journal, no-no. https://doi.org/10.1111/j.1365-313x.2012.04949.x