THCAS and CBDAS have been more studied and characterized than CBCAS, although CBCAS’s high sequence similarity with THCAS suggests it shares similar biological activities.With regards to localization and expression in the Cannabis sativa plant, CBDAS and THCAS have been demonstrated to be catalytically active in the glandular trichome’s extracellular cavity and need FAD as well as oxygen in order to functionally express.In contrast, the CBCAS enzyme is not oxygen dependent and can also be inhibited by hydrogen peroxide.As mentioned, THCAS and CBCAS share a 92% sequence similarity with each other, whereas THCAS and CBDAS are 84% identical to each other and CBCAS and CBDAS are 83% identical to each other; therefore, all three cyclase enzymes have high sequence similarity with each other. Sequence analysis of variants of the cyclase enzymes indicated that CBDA is likely the ancestral enzyme from which THCAS evolved from. Further sequence analysis of THCAS showed a flavinylation consensus sequence with His114 being the likely FAD-binding site, exhibiting similarity to CBDAS in this regard.Since both CBDAS and THCAS are flavinated enzymes, it is postulated that they have the same reaction mechanism. With regards to CBCAS, although much is unknown, it has been reported that based on its kinetic data , CBCAS has higher affinity for CBGA than both THCAS and CBDAS. These oxidocyclase enzymes are only capable of enzymatically acting up CBGA, not the decarboxylated CBG product.Due to the reported potent biological activity of THCP, the heptyl analog of THC,grow racks we determined to focus on achieving function expression of THCAS in our platform since our novel platform produces the heptyl variant of olivetolic acid.
As previously described, THCP was isolated from the Cannabis plant in small quantities in 2019 with pharmacological data showing that THCP has a Ki to CB1 of 1.2 mM, and a Ki to CB2 of 6.2 mM, ~ 30 and 6 times more effective in binding than THC, respectively.102 Even the THCA molecule, although unlike its decarboxylated form THC, is not psychoactive, has been investigated for its neuroprotective, anti-neoplastic, immunomodulator, and anti-inflammatory effects further making the pursuit and optimization of in vivo functional expression of THCAS a priority. Different groups have reported of engineering strains from Saccharomyces cerevisiae and Komagataella phaffi capable of functional expression of THCAS. Zirpel et al. demonstrated through the engineering of THCAS, optimization of culturing conditions, and overexpression of helper proteins, 83% conversion of CBGA to THCA in Komagataella phaffi. Zirpel et al were first able to obtain functional expression of THCAS in both S. cerevisiae and K. phaffi by utilizing a signal peptide from the vacuolar proteinase, proteinase A. The group cleaved the 28 amino acid N-terminal plastid signal peptide from the THCAS and inserted the proteinase A signal peptide. Additionally, they knocked out the proteinase A gene in both strains, responsible for degrading genes targeted to the vacuole. With functional expression in both yeast strains achieved through this process, they sought to optimize culturing conditions.The group assayed the strains at different temperatures and times and observed that the highest intracellular activity of THCAS was achieved at 15C for 192 hours. The temperature of the expression was the key change from other groups which had expressed THCAS at 30C and 37C. The expression of THCAS at 15 C increased volumetric THCAS by 6350%. Additionally concerning culturing conditions, Zirpel et al observed that addition of casamino acids, biotin, riboflavin, and yeast nitrogen base further increased THCAS functional expression.
Lastly, the overexpression of ER chaperones, Kar2p, CNE1p, the Pd1p foldase, the unfolded protein activator , as well as the FAD synthetase increased functional expression of THACS in K. phaffi, with the expression of HAC1p, having the greatest impact, increasing functional expression 4-fold.Through implementation of these concepts, we achieved functional expression of THCAS in our Saccharomyces cerevisiae strain. We already had obtained a S. cerevisiae strain with the pep4 knockout, so with transformation of the THCAS gene with the proA signal and with feeding CBGA, we observed production of THCA in YPD media at 30C. To increase functional expression, we added 13.8g/L yeast nitrogen base and 5 g/L casamino acids into the YPD media and observed a slight increase in production. To further increase expression, we overexpressed the HAC1p with the THCAS in our S. cerevisiae strain and observed a notable increase in production. We also then tested the temperature similar to Zirpel et al. The initial THCAS functional expression assays were done in media cultured at 30C. We therefore tested the temperature in a few different ways: 1) Culturing the seed culture and growth culture at 15C and feeding CBGA at day 2 2) Culturing the seed culture and growth culture at 30C and feeding CBGA at day 2 3) Culturing the seed culture and growth culture at 30C for 5 days and feeding CBGA at day 6. 4) Culturing the seed culture and growth culture at 15C for 5 days. At the 6th day, feed CBGA and culture at 30C 5) Culturing the seed culture and growth culture at 15C for 5 days and feeding CBGA at day 6. As the data indicates, option 4 had the greatest increase in functional THCAS expression, achieving greater than 50% conversion of CBGA to THCA based on UV area under the curve, a significant increase over the 3% estimated conversion rate when we initially expressed THCAS in S. cerevisiae.
In conclusion, we have demonstrated that through genome mining of fungal bio-synthetic gene clusters, we were able to identify a cluster in Metarhizium anisopliae that produces olivetolic acid, the first key intermediate in the cannabinoid bio-synthetic pathway as well as unsaturated olivetolic acid, sphaerophorolcarboxylic acid, and unsaturated sphaerophorolcarboxylic acid at moderate to high titers. We also identified homologous clusters that produced these same compounds as well as a homologous cluster found in Talaromyces islandicus that selectively produces olivetolic acid. From there, we desired to engineer our platform in three different aspects: i) by increasing the titer ii) by increasing the diversity our product profile iii) by going downstream to achieve the final elaborated cannabinoids.To increase the titer, we utilized two different approaches. In one, we utilized JMP software to develop a design of experiments approach to increase titer by optimizing the culture media. We initially screened for nine different components in the media, testing to determine which components had statistically significant effects on the titer production. We identified three components which we then optimized for using a response surface methodology approach. Using this approach, we were able to identify a culturing media that assists us in reaching titers almost 2-fold more than our original CD-ST culturing media. In our second approach to increasing the titer of our platform, we increased the malonyl-CoA pool in our model Aspergillus nidulans host by overexpressing the acetyl-CoA carboxylase gene, the enzyme responsible for converting acetyl-CoA to malonyl-CoA. Overexpressing of acetyl-CoA carboxylase helped us achieve greater than 2.5-fold increase in titer. We also desired to increase the diversity of our product profile. To do so, we also employed two different approaches. In one, we made mutations to the ketosynthase domain of our HRPKS, Ma_OvaA. The KS domain is responsible for decarboxylative Claisen condensation, extending the carbon-carbon chain length. We made mutations in the KS domain both manually and through the use of the automated Tecan liquid handling system integrated with a ThermoFisher Momentum Laboratory Automation System. We were able to identify a couple of mutations that produced different products from our original platform including divarinic acid as well as the olivetolic acid nonyl variant,grow table undecyl variant, and ethyl variant. We also further genome mining to identify more homologous clusters to our original platform. Utilizing our in-house strain database, we were able to identify a cluster in Penicillium thomii r89 that when heterologously expressed in Aspergillus nidulans, produced a nonyl variant of olivetolic acid with two degrees of unsaturation and a heptyl variant containing one degree ofunsaturation as well as a hydroxy group. Therefore, both methods were effective in producing more olivetolic acid variants and there is still much potential in discovering more variants with regards to these methods since especially with regards to the genome mining aspect, there is still much unexplored space. Lastly, we attempted to go downstream of the pathway to achieve the final elaborated cannabinoids. To do so, we mined for different prenyltransferase enzymes capable of geranylating olivetolic acid to form cannabigerolic acid. We were able to identify one in Talaromyces islandicus capable of geranylating olivetolic acid to form cannabigerolic acid in Aspergillus nidulans, giving us a platform where through the heterologous expression of four fungal genes, we were able to produce the first cannabinoid, CBGA. Utilizing CsPT4, we were also able to achieve de novo production of the heptyl variant of CBGA, when we expressed the Ma_OvA genes with CsPT4 in Saccharomyces cerevisiae. We also were able to achieve functional expression of THCAS in S. cerevisiae and were able to greatly increase expression by optimization of media, over expression of helper proteins, and culturing at lower temperatures.
Therefore, we have developed a different method for microbial production of cannabinoids, one that does not rely heavily on the cannabis plant genes and has potential to produce rare and new to-nature cannabinoids.With its roots in international treaties signed during the League of Nations Era, the transnational legal order of cannabis prohibition represents one of the most sustained efforts to develop internationally applicable standards for governing illicit markets.The vast majority of United Nations member states are now parties to the three major international drug conventions, which require criminalizing the production, distribution, and use of cannabis. Over the past decades, the cannabis prohibition TLO has come to encompass an extensive array of legal instruments for monitoring implementation efforts,disseminating information on the activities of drug trafficking networks,and facilitating cooperation among national police forces.However, despite the extensive institutionalization of this TLO, cannabis remains the most widely used illegal drug in the world. The 2018 World Drug Report estimates that at least 192 million people aged 15–64 had used cannabis in the preceding year.With the percentage of adults reporting cannabis use in North American and European countries far exceeding the international average, cannabis use has become integrated into mainstream culture in a large number of countries.In an era that is often characterized as one of a growing isomorphism of the laws and procedures governing criminal activities in different countries,the issue area of cannabis policy undergoes processes of fragmentation and polarization.Some countries continue to criminalize all forms of medical and recreational uses of cannabis. Others have sought to “separate the market” for cannabis from that of other drugs by decriminalizing the possession of small amounts of marijuana, authorizing its use for medical purposes, and establishing administrative measures for taxing and regulating the commercial sale of the drug.These reforms have gained international momentum despite resistance from key actors in the international drug control system, including the International Narcotic Control Board and the US federal government.The proliferation of cannabis liberalization reform is frequently depicted as a historical step toward the collapse not only of this TLO but of the entire edifice of the international narcotic control system of which it forms a part.How deep is the current crisis of the cannabis prohibition TLO? What are its causes and consequences? What does this case study reveal about the conditions under which criminal justice TLOs rise and fall? In this Article, I explore these questions to demonstrate the complex ways in which the cannabis prohibition TLO has served as a battleground between competing conceptions of the role of criminal law in addressing social and medical harms. Drawing on TLO theory,the Article shows that the capacity of the cannabis prohibition TLO to regulate the practices of legal actors at the international, national, and local levels has been eroded as a result of effective contestations of the input and output legitimacy of its governance endeavors. The rapid and widespread diffusion of new models of decriminalization, depenalization, and legalization has relied on the operation of mechanisms of recursive transnational lawmaking. These mechanisms originate from the indeterminacy of drug prohibition norms, the ideological contradictions between competing interpretations of their meaning, the impact of diagnostic struggles over the social issues that the international drug control system should address, and the mismatch between the actors shaping formal prohibition norms at the international level and those implementing these norms in national and local contexts.