Cannabinoids used in Pain Management
January 21, 2022
Cannabis sativa and its use in the treatment of Autistic Spectrum Disorder
February 4, 2022Scientific information for healthcare professionals
Author : Dr.Elmo Resende, Ph.D
| Director of R&D - Piauhy Labs
Cannabinoid Biosynthesis | Definition
Biotransformations consist of chemical conversions of a substance (starting material) into a product, using whole cells of plants, microorganisms and animals, containing the necessary enzyme(s), or through enzymes isolated.
Reactions involving microorganisms are carried out using pure cultures of the desired microorganism, grown in specific culture media, containing the nutrients necessary for their growth.
Many bioreactions are carried out using enzymes. These follow a classification created by the International Union of Biochemistry, which divides them into six main classes, namely: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
Several parameters need to be considered for a reaction using isolated enzymes, such as the type of reaction desired, the solubility of the substrate, the activity of the enzyme in question and the occurrence of parallel reactions. Often, there is also the need to use special techniques for immobilizing these enzymes due to their instability, given the operating conditions used, the economic need for reuse of them and their low tolerance in the face of high substrate concentrations.
Biotransformation | Reactions
Biotransformations can be made with natural or synthetic substrates, and it is possible to use a wide variety of substances as substrates for reactions, such as terpenes, steroids and alkaloids, among others. These can be used both to obtain new products and also known products, in a more efficient way.
Among the advantages of these reactions are the possibility of obtaining regio, stereo and enantioselectivities, different from those obtained in classical organic synthesis, the mild conditions used in them, such as pH around 7 and room temperature, and the possibility of functionalizing positions of low reactivity in molecules used as substrates, in places where the application of conventional chemistry is difficult.
Biotransformations involving whole cells of microorganisms are carried out in aqueous systems, as these are compatible with cell growth. Biotransformation reactions with fungi are important allies in organic, industrial or academic synthesis. They are used to add value to simpler substances, such as in the synthesis of drugs such as vitamins, steroids, alkaloids and antibiotics. The application of biotransformations in fine chemistry is very cost-effective.
Cannabis sativa L. is an annual wind-pollinated herb, usually dioecious, with male and female flowers growing on separate plants. This plant is well known for the biosynthesis of cannabinoids, the terpenophenolic constituents that have psychoactive effects. But since other plants also have secondary metabolites that interact with human cannabinoid receptors, a new definition had to be made. Therefore, phytocannabinoids are now defined as any natural plant-derived compounds that can act as a ligand for human cannabinoid receptors (CB1 and CB2) or that share chemical similarity with cannabinoids. Interestingly, all parts of the Cannabis plant, with the exception of the seeds, may contain cannabinoids, but mostly they accumulate in the glandular trichomes of female flowers.
Therefore, a biotechnological approach transferring enzymes from the plant biosynthetic pathway from Δ9-tetrahydrocannabinolic acid (THCA), the precursor of THC, to a microbial production host may represent an appropriate alternative. The enzyme Δ9-tetrahydrocannabinolic acid synthase (THCAS), which catalyzes the last step of THCA biosynthesis, and the oxidative cyclization of cannabigerolic acid (CBGA) were expressed in low amounts in recombinant tobacco hair roots, insect cell cultures and secretion of pastoral cultures Pichia (Komagataella).
The heterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Products Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned in Saccharomyces cerevisiae, heterologous biosynthesis has been fully incorporated as a proof of concept. The low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of the recombinant catalyst by protein engineering and consequent improvement of the yeast chassis C-flow for sufficient supply of precursor (acetyl-CoA), energy (ATP) and of NADH.
Cannabinoids appear to be a unique class of natural by-products limited to Cannabis sativa L. Recently, prenylated olivetolic acid derivatives and other structurally related prenylated phenolics have also been identified in several genera and species such as Helichrysum umbraculigerum Less or liverwort Radula marginata Taylor. The biosynthesis of tetrahydrocannabinolic acid (THCA) and its precursors are derived from the mevalonate and olivetolic acid pathway, as we understand it today. Without going into the details of molecular biology, genetics and spatial resolution of biosynthesis, all biosynthetic enzymes compromised on the way to tetrahydrocannabinolic acid, but also cannabidiolic acid and cannabichromenic acid, share the precursor cannabigerolic acid (CBGA). Furthermore, all conversion products have the same mass and only differ structurally.
Biotechnological studies of systems are conducted with S. cerevisiae as a model organism. This yeast is widely used in industry because of its GRAS status, the physiological properties are easy to handle and there is extensive knowledge of its genomics, proteomics and other related fields of “OMICS”. It is very interesting for THCA production because of the biosynthesis of high-throughput terpenoids and the delivery of IPP/DMAPP.
For the biosynthesis and production of cannabinoids, S. cerevisiae has several advantages: first, a greater biocatalytic activity explained by the low concentration of poorly folded biosynthetic proteins and the absence of inactive inclusion bodies; second, THCAS glycosylation resulting in better solubility correlated with increased biocatalytic rate; and, finally, the ability to secrete cannabinoids to minimize the risk of auto-toxication and allow for easier downstreaming and isolation.
Acetyl-CoA is the committed precursor to the pathway from mevalonate and olivetolic acid to GPP and olivetolic acid. The regulation and increase of acetyl-CoA or short pool of C2 is essential for renewal and catalytic performance. Consequently, intracellular levels of acetyl-CoA, as well as its delivery from the primary pathway in S. cerevisiae for the biosynthesis of heterologous cannabinoids, are a significant concern in any successful bioengineering strategy.
The efficiency of heterologous cannabinoid biosynthesis depends on the supply of olivetolic acid, which is, together with GPP, consumed in equimolar amounts by NphB forming CBGA.
The production of geranyl diphosphate (GPP) via the mevalonate pathway has significant potential for metabolic engineering approaches. It presents several enzymes with remarkably low conversion rates and concludes in an unfavorable ratio of farnesyl diphosphate (FPP) to geranyl diphosphate (GPP).
After the supply of olivetolic acid and GPP, the last two enzymatic catalytic steps towards THCA are the biotransformation to CBGA by a prenyltransferase and the subsequent conversion to THCA by THCA synthase (THCAS).
In recent years, several processes have been developed using S. cerevisiae as a host organism for the production of compounds such as vanillin, amorphous-4,11-diene or artemisinic acid, indicating that yeast has significant potential as an industrial platform host for large-scale production. To increase THCA yields and translate laboratory scale production to industrial process scale, process parameters and growing conditions need to be optimized to keep costs down.
Terpenoids, also known as isoprenoids, are a broad and diverse class of natural plant products with significant industrial and pharmaceutical importance. Many of these natural products have antitumor, anti-inflammatory, antibacterial, antiviral and antimalarial effects, support transdermal absorption, prevent and treat cardiovascular disease, and have hypoglycemic activities. The production of these compounds is usually carried out through extraction from their natural sources or chemical synthesis.
However, these processes are generally unsustainable, produce low yields and result in the waste of substantial resources, most of which are limited. Microbial production of terpenoids offers a sustainable and environmentally friendly alternative.
In recent years, the yeast Saccharomyces cerevisiae has become a suitable cell factory for the biosynthesis of industrial terpenoids due to developments in omics studies (genomics, transcriptomics, metabolomics, proteomics) and mathematical modeling. Furthermore, fermentation development is of significant importance in obtaining high titer, yield, and productivity (TRP) of these compounds.
Detailed transcriptomics performed on extractions of glandular trichomes as well as female floral tissues of Cannabis sativa at various stages of development indicate that candidate genes involved in terpene and cannabinoid synthesis are abundantly expressed in trichomes. A recent study acquired high quality glandular trichome transcriptomes from nine different commercial Cannabis strains in an attempt to characterize the terpene synthases. A phylogenetic analysis of the entire Cannabis sativa genome contig database also revealed 24 O-methyltransferases and eight putative aromatic prenyltransferases, one of which was characterized as the penultimate synthase for cannaflavins A and B.
The use of microbial biosynthesis for the production of economically relevant terpenoids limits the need to grow, harvest and extract plant material. This provides an ecological synthesis platform for specialized terpenoids and allows their production in high concentration and purity. Advances in technologies and strategies for the identification and heterologous expression of terpenoid biosynthesis pathways in microorganisms will provide numerous opportunities for future research. Although there has been recent success in engineering prokaryotes to produce terpenes, yeast will prove to be the ideal production host for more complex terpenoid derivatives and should be the basis for future efforts.
In addition to the psychoactive Δ9-tetrahydrocannabinol (THC) and pharmacologically related cannabinoids (e.g., Cannabidiol - CBD) that normally accumulate in a variety of Cannabis sativa cultivars, there are a multitude of specialized metabolites in this plant species believed to contribute to its drug potential. One of these classes of compounds are the prenylated flavonoids known as canflavins A and B.
The interest around canflavins A and B in the Cannabis community stems from three seminal studies: In the first, the researchers demonstrated that free THC and CBD extracts of Cannabis sativa could reduce the cataleptic effects of THC in mice and that this effect could be reversed by administration of prostaglandin E2 (PGE2). The researchers reportedly identified the causative agent in these extracts as canflavins A and B and found that these prenylated flavonoids could inhibit PGE2 production in human rheumatoid synovial cells and provide anti-inflammatory benefits that were approximately thirty times more effective than aspirin. It was later shown that the underlying basis for their potent anti-inflammatory properties was that cannflavins A and B act to inhibit the in vivo production of two pro-inflammatory mediators, prostaglandin E2 and leukotrienes.
Commercial development of a cell-free system faces challenges; however, the Merck company recently reported the commercial development of a new nine-enzyme cascade. In one study, the implementation of a 12-enzyme system was suggested, which has many advantages that make it interesting for potential commercial development. It uses twelve enzymes, employs inexpensive starting materials, potentially allows for aerobic or anaerobic operation as there are no oxygen-requiring steps and is highly modular. In this method, OA and DA substances are produced in situ, starting from low-cost organic acids.
Engineering microorganisms to produce valuable molecules is a sustainable alternative to conventional production methods, already providing a solution for the production of numerous compounds totaling a $230 billion global market. However, achieving economically viable productivity is still a major obstacle in the process of achieving industrial production of various valuable compounds.
In another study, the biosynthetic pathway of cannabinoid production via genetically modified brewer's yeast (S. cerevisiae) was studied. The genes that code for specific enzymes were taken from organisms of different origins and inserted into the yeast genome, in addition to using the own enzymes existing in the organisms. Yeast converts sugars and hexanoic acid into the component parts and assembles the cannabinoid precursor molecule, which is then broken down into different cannabinoids via different enzymes.
Cannabis sativa is a treasure trove of specialized metabolites with an emphasis on phytocannabinoids. Due to the biological activities of cannabinoids, including psychoactive ones, humans have used cannabis extensively as a medicinal plant in the treatment of pain, nausea, glaucoma and other illnesses. Today, legally-derived cannabis products represent a fast-growing global industry expected to reach $57 billion by 2027, with pharmaceutical cannabinoid consumption accounting for one-third of total profit.
Cannabinoid formation requires precursors of two biosynthetic pathways, the polyketide and methylerythritol phosphate isoprenoid (MEP) biosynthetic pathways. Hexanoic acid is converted to olivetolic acid by the action of acyl activating enzyme 1 (AAE1), olivetol synthase (OLS) and olivetolic acid cyclase (OAC). Olivetolic acid (OA) is then prenylated with geranyl diphosphate (GPP) derived from the MEP plastid pathway as a donor in a reaction catalyzed by a geranyldiphosphate: olivetolate geranyltransferase (or cannabigerolic acid synthase, CBGAS). The cannabigerolic acid (CBGA) formed represents the first genuine cannabinoid in the biosynthetic pathway. CBGA functions as a substrate for the oxidocyclases Δ9-tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS) and cannabichromene acid synthase (CBCAS), forming Δ9 - tetrahydrocannabinolic acid, cannabidiolic acid, and cannabichromenic acid, respectively.
In fact, biosynthesis is the production of molecules using living cells such as bacteria, yeast or algae. The company Piauhy Labs intends to introduce the genes of the cannabinoid biosynthetic pathway (obtained from the Cannabis plant) in the genome of capable cells (bacteria), in order to produce all the cannabinoids, starting from primary sugars. Then, from these genetically modified cell lines, it is suggested to build a highly reproducible platform, with high clinical purity, for the scalable synthesis of cannabinoids that will be used for therapeutic purposes, in particular therapies directed to pathologies of the central nervous system.
That said, the first step in this process involves inserting a biosynthetic cluster (a physically grouped group of two or more genes in a specific genome that together encode a biosynthetic pathway for the production of a specialized metabolite) into the DNA vector. Then follows the genomic engineering of the host; here, the DNA is inserted into the bacteria, where it provides the instructions for making cannabinoid components. Finally, the process is conducted on a large scale, which results in the production of materials that can be further processed into purified cannabinoids.
Piauhy Labs intends to obtain a Certification of Compliance with Good Laboratory Practices, in accordance with the OECD principles, for the pharmaceutical area, so that the results obtained from its research are properly used for the granting of licenses or for the registration of pharmaceutical products, including medicines for human use and similar products.
As a result of the intended research processes, Piauhy Labs wants to create patents and originate intellectual property, with the ultimate goal of producing innovative medicines, medicines that improve the quality of life of patients suffering from various diseases, notably diseases related to the central nervous system.
Elmo Resende, Ph.D
Director of R&D
Piauhy Labs
References
Belcher, M. S.; Mahinthakumar, J. and Keasling, J. D. New frontiers: harnessing pivotal advances in microbial engineering for the biosynthesis of plant-derived terpenoids. Current Opinion in Biotechnology 65, 88–93, 2020.
Braich, S.; Baillie, R. C.; Jewell, L. S.; Spangenberg, G. C. and Cogan, N. O. I. Generation of a Comprehensive Transcriptome Atlas and Transcriptome Dynamics in Medicinal Cannabis. Scientific Reports 9 (1), 16583, 2019.
Carsanba, E.; Pintado, M. and Oliveira, C. Fermentation Strategies for Production of Pharmaceutical Terpenoids in Engineered Yeast. Pharmaceuticals (Basel) 14 (4), 295, 2021.
Degenhardt, F.; Stehle, F. and Kayser, O. Handbook of Cannabis and Related Pathologies. Biology, Pharmacology, Diagnosis, and Treatment. Chapter 2 - The Biosynthesis of Cannabinoids 13-23, 2017.
Dusséaux, S.; Wajn, W. T.; Liu, Y.; Ignea, C. and Kampranis, S. C. Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proc. Natl. Acad. Sci. USA Dec 15, 117 (50), 31789-31799, 2020.
Gülck, T.; Booth, J. K.; Carvalho, Â.; Khakimov, B.; Crocoll, C.; Motawia, M. S.; Møller, B. L.; Bohlmann, J. and Gallage, N. J. Synthetic Biology of Cannabinoids and Cannabinoid Glucosides in Nicotiana benthamiana and Saccharomyces cerevisiae. J. Nat. Prod. 23, 83 (10), 2877-2893, 2020.
Rea, K. A.; Casaretto, J. A.; Al-Abdul-Wahid, M. S.; Sukumaran, A.; Geddes-McAlister, J.; Rothstein, S. J. and Akhtar, T. A. Biosynthesis of cannflavins A and B from Cannabis sativa L. Phytochemistry 164, 162–171, 2019.
Smith, M. "Biosynthesis of Cannabinoids in S. cerevisiae". Thinking Matters Symposium. 82. 2020.
Thomas, F.; Schmidt, C. and Kayser, O. Bioengineering studies and pathway modeling of the heterologous biosynthesis of tetrahydrocannabinolic acid in yeast. Applied Microbiology and Biotechnology 104, 9551–9563, 2020.
Valliere, M. A.; Korman, T. P.; Arbing, M. A. and Bowie, J. U. A bio-inspired cell-free system for cannabinoid production from inexpensive inputs. Nat Chem Biol. 16 (12), 1427-1433, 2020.
Xiaozhou, L.; Reiter, M. A.; d’Espaux, L.; Wong, J.; 3,12, Denby, C. M. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature Mar, 567 (7746), 123-126, 2019.
Zirpel. B.; Stehle, F. and Kayser, O. Production of Δ9-Tetrahydrocannabinolic acid from Cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-Tetrahydrocannabinolic acid synthase from Cannabis sativa L. Biotechnol. Lett. 37 (9), 1869-75, 2015.
Cannabinoid Biosynthesis | Definition
Biotransformations consist of chemical conversions of a substance (starting material) into a product, using whole cells of plants, microorganisms and animals, containing the necessary enzyme(s), or through enzymes isolated.
Reactions involving microorganisms are carried out using pure cultures of the desired microorganism, grown in specific culture media, containing the nutrients necessary for their growth.
Many bioreactions are carried out using enzymes. These follow a classification created by the International Union of Biochemistry, which divides them into six main classes, namely: oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases.
Several parameters need to be considered for a reaction using isolated enzymes, such as the type of reaction desired, the solubility of the substrate, the activity of the enzyme in question and the occurrence of parallel reactions. Often, there is also the need to use special techniques for immobilizing these enzymes due to their instability, given the operating conditions used, the economic need for reuse of them and their low tolerance in the face of high substrate concentrations.
Biotransformation | Reactions
Biotransformations can be made with natural or synthetic substrates, and it is possible to use a wide variety of substances as substrates for reactions, such as terpenes, steroids and alkaloids, among others. These can be used both to obtain new products and also known products, in a more efficient way.
Among the advantages of these reactions are the possibility of obtaining regio, stereo and enantioselectivities, different from those obtained in classical organic synthesis, the mild conditions used in them, such as pH around 7 and room temperature, and the possibility of functionalizing positions of low reactivity in molecules used as substrates, in places where the application of conventional chemistry is difficult.
Biotransformations involving whole cells of microorganisms are carried out in aqueous systems, as these are compatible with cell growth. Biotransformation reactions with fungi are important allies in organic, industrial or academic synthesis. They are used to add value to simpler substances, such as in the synthesis of drugs such as vitamins, steroids, alkaloids and antibiotics. The application of biotransformations in fine chemistry is very cost-effective.
Cannabis sativa L. is an annual wind-pollinated herb, usually dioecious, with male and female flowers growing on separate plants. This plant is well known for the biosynthesis of cannabinoids, the terpenophenolic constituents that have psychoactive effects. But since other plants also have secondary metabolites that interact with human cannabinoid receptors, a new definition had to be made. Therefore, phytocannabinoids are now defined as any natural plant-derived compounds that can act as a ligand for human cannabinoid receptors (CB1 and CB2) or that share chemical similarity with cannabinoids. Interestingly, all parts of the Cannabis plant, with the exception of the seeds, may contain cannabinoids, but mostly they accumulate in the glandular trichomes of female flowers.
Therefore, a biotechnological approach transferring enzymes from the plant biosynthetic pathway from Δ9-tetrahydrocannabinolic acid (THCA), the precursor of THC, to a microbial production host may represent an appropriate alternative. The enzyme Δ9-tetrahydrocannabinolic acid synthase (THCAS), which catalyzes the last step of THCA biosynthesis, and the oxidative cyclization of cannabigerolic acid (CBGA) were expressed in low amounts in recombinant tobacco hair roots, insect cell cultures and secretion of pastoral cultures Pichia (Komagataella).
The heterologous biosynthesis of tetrahydrocannabinolic acid (THCA) in yeast is a biotechnological process in Natural Products Biotechnology that was recently introduced. Based on heterologous genes from Cannabis sativa and Streptomyces spp. cloned in Saccharomyces cerevisiae, heterologous biosynthesis has been fully incorporated as a proof of concept. The low titer and insufficient biocatalytic rate of most enzymes require systematic optimization of the recombinant catalyst by protein engineering and consequent improvement of the yeast chassis C-flow for sufficient supply of precursor (acetyl-CoA), energy (ATP) and of NADH.
Cannabinoids appear to be a unique class of natural by-products limited to Cannabis sativa L. Recently, prenylated olivetolic acid derivatives and other structurally related prenylated phenolics have also been identified in several genera and species such as Helichrysum umbraculigerum Less or liverwort Radula marginata Taylor. The biosynthesis of tetrahydrocannabinolic acid (THCA) and its precursors are derived from the mevalonate and olivetolic acid pathway, as we understand it today. Without going into the details of molecular biology, genetics and spatial resolution of biosynthesis, all biosynthetic enzymes compromised on the way to tetrahydrocannabinolic acid, but also cannabidiolic acid and cannabichromenic acid, share the precursor cannabigerolic acid (CBGA). Furthermore, all conversion products have the same mass and only differ structurally.
Biotechnological studies of systems are conducted with S. cerevisiae as a model organism. This yeast is widely used in industry because of its GRAS status, the physiological properties are easy to handle and there is extensive knowledge of its genomics, proteomics and other related fields of “OMICS”. It is very interesting for THCA production because of the biosynthesis of high-throughput terpenoids and the delivery of IPP/DMAPP.
For the biosynthesis and production of cannabinoids, S. cerevisiae has several advantages: first, a greater biocatalytic activity explained by the low concentration of poorly folded biosynthetic proteins and the absence of inactive inclusion bodies; second, THCAS glycosylation resulting in better solubility correlated with increased biocatalytic rate; and, finally, the ability to secrete cannabinoids to minimize the risk of auto-toxication and allow for easier downstreaming and isolation.
Acetyl-CoA is the committed precursor to the pathway from mevalonate and olivetolic acid to GPP and olivetolic acid. The regulation and increase of acetyl-CoA or short pool of C2 is essential for renewal and catalytic performance. Consequently, intracellular levels of acetyl-CoA, as well as its delivery from the primary pathway in S. cerevisiae for the biosynthesis of heterologous cannabinoids, are a significant concern in any successful bioengineering strategy.
The efficiency of heterologous cannabinoid biosynthesis depends on the supply of olivetolic acid, which is, together with GPP, consumed in equimolar amounts by NphB forming CBGA.
The production of geranyl diphosphate (GPP) via the mevalonate pathway has significant potential for metabolic engineering approaches. It presents several enzymes with remarkably low conversion rates and concludes in an unfavorable ratio of farnesyl diphosphate (FPP) to geranyl diphosphate (GPP).
After the supply of olivetolic acid and GPP, the last two enzymatic catalytic steps towards THCA are the biotransformation to CBGA by a prenyltransferase and the subsequent conversion to THCA by THCA synthase (THCAS).
In recent years, several processes have been developed using S. cerevisiae as a host organism for the production of compounds such as vanillin, amorphous-4,11-diene or artemisinic acid, indicating that yeast has significant potential as an industrial platform host for large-scale production. To increase THCA yields and translate laboratory scale production to industrial process scale, process parameters and growing conditions need to be optimized to keep costs down.
Terpenoids, also known as isoprenoids, are a broad and diverse class of natural plant products with significant industrial and pharmaceutical importance. Many of these natural products have antitumor, anti-inflammatory, antibacterial, antiviral and antimalarial effects, support transdermal absorption, prevent and treat cardiovascular disease, and have hypoglycemic activities. The production of these compounds is usually carried out through extraction from their natural sources or chemical synthesis.
However, these processes are generally unsustainable, produce low yields and result in the waste of substantial resources, most of which are limited. Microbial production of terpenoids offers a sustainable and environmentally friendly alternative.
In recent years, the yeast Saccharomyces cerevisiae has become a suitable cell factory for the biosynthesis of industrial terpenoids due to developments in omics studies (genomics, transcriptomics, metabolomics, proteomics) and mathematical modeling. Furthermore, fermentation development is of significant importance in obtaining high titer, yield, and productivity (TRP) of these compounds.
Detailed transcriptomics performed on extractions of glandular trichomes as well as female floral tissues of Cannabis sativa at various stages of development indicate that candidate genes involved in terpene and cannabinoid synthesis are abundantly expressed in trichomes. A recent study acquired high quality glandular trichome transcriptomes from nine different commercial Cannabis strains in an attempt to characterize the terpene synthases. A phylogenetic analysis of the entire Cannabis sativa genome contig database also revealed 24 O-methyltransferases and eight putative aromatic prenyltransferases, one of which was characterized as the penultimate synthase for cannaflavins A and B.
The use of microbial biosynthesis for the production of economically relevant terpenoids limits the need to grow, harvest and extract plant material. This provides an ecological synthesis platform for specialized terpenoids and allows their production in high concentration and purity. Advances in technologies and strategies for the identification and heterologous expression of terpenoid biosynthesis pathways in microorganisms will provide numerous opportunities for future research. Although there has been recent success in engineering prokaryotes to produce terpenes, yeast will prove to be the ideal production host for more complex terpenoid derivatives and should be the basis for future efforts.
In addition to the psychoactive Δ9-tetrahydrocannabinol (THC) and pharmacologically related cannabinoids (e.g., Cannabidiol - CBD) that normally accumulate in a variety of Cannabis sativa cultivars, there are a multitude of specialized metabolites in this plant species believed to contribute to its drug potential. One of these classes of compounds are the prenylated flavonoids known as canflavins A and B.
The interest around canflavins A and B in the Cannabis community stems from three seminal studies: In the first, the researchers demonstrated that free THC and CBD extracts of Cannabis sativa could reduce the cataleptic effects of THC in mice and that this effect could be reversed by administration of prostaglandin E2 (PGE2). The researchers reportedly identified the causative agent in these extracts as canflavins A and B and found that these prenylated flavonoids could inhibit PGE2 production in human rheumatoid synovial cells and provide anti-inflammatory benefits that were approximately thirty times more effective than aspirin. It was later shown that the underlying basis for their potent anti-inflammatory properties was that cannflavins A and B act to inhibit the in vivo production of two pro-inflammatory mediators, prostaglandin E2 and leukotrienes.
Commercial development of a cell-free system faces challenges; however, the Merck company recently reported the commercial development of a new nine-enzyme cascade. In one study, the implementation of a 12-enzyme system was suggested, which has many advantages that make it interesting for potential commercial development. It uses twelve enzymes, employs inexpensive starting materials, potentially allows for aerobic or anaerobic operation as there are no oxygen-requiring steps and is highly modular. In this method, OA and DA substances are produced in situ, starting from low-cost organic acids.
Engineering microorganisms to produce valuable molecules is a sustainable alternative to conventional production methods, already providing a solution for the production of numerous compounds totaling a $230 billion global market. However, achieving economically viable productivity is still a major obstacle in the process of achieving industrial production of various valuable compounds.
In another study, the biosynthetic pathway of cannabinoid production via genetically modified brewer's yeast (S. cerevisiae) was studied. The genes that code for specific enzymes were taken from organisms of different origins and inserted into the yeast genome, in addition to using the own enzymes existing in the organisms. Yeast converts sugars and hexanoic acid into the component parts and assembles the cannabinoid precursor molecule, which is then broken down into different cannabinoids via different enzymes.
Cannabis sativa is a treasure trove of specialized metabolites with an emphasis on phytocannabinoids. Due to the biological activities of cannabinoids, including psychoactive ones, humans have used cannabis extensively as a medicinal plant in the treatment of pain, nausea, glaucoma and other illnesses. Today, legally-derived cannabis products represent a fast-growing global industry expected to reach $57 billion by 2027, with pharmaceutical cannabinoid consumption accounting for one-third of total profit.
Cannabinoid formation requires precursors of two biosynthetic pathways, the polyketide and methylerythritol phosphate isoprenoid (MEP) biosynthetic pathways. Hexanoic acid is converted to olivetolic acid by the action of acyl activating enzyme 1 (AAE1), olivetol synthase (OLS) and olivetolic acid cyclase (OAC). Olivetolic acid (OA) is then prenylated with geranyl diphosphate (GPP) derived from the MEP plastid pathway as a donor in a reaction catalyzed by a geranyldiphosphate: olivetolate geranyltransferase (or cannabigerolic acid synthase, CBGAS). The cannabigerolic acid (CBGA) formed represents the first genuine cannabinoid in the biosynthetic pathway. CBGA functions as a substrate for the oxidocyclases Δ9-tetrahydrocannabinolic acid synthase (THCAS), cannabidiolic acid synthase (CBDAS) and cannabichromene acid synthase (CBCAS), forming Δ9 - tetrahydrocannabinolic acid, cannabidiolic acid, and cannabichromenic acid, respectively.
In fact, biosynthesis is the production of molecules using living cells such as bacteria, yeast or algae. The company Piauhy Labs intends to introduce the genes of the cannabinoid biosynthetic pathway (obtained from the Cannabis plant) in the genome of capable cells (bacteria), in order to produce all the cannabinoids, starting from primary sugars. Then, from these genetically modified cell lines, it is suggested to build a highly reproducible platform, with high clinical purity, for the scalable synthesis of cannabinoids that will be used for therapeutic purposes, in particular therapies directed to pathologies of the central nervous system.
That said, the first step in this process involves inserting a biosynthetic cluster (a physically grouped group of two or more genes in a specific genome that together encode a biosynthetic pathway for the production of a specialized metabolite) into the DNA vector. Then follows the genomic engineering of the host; here, the DNA is inserted into the bacteria, where it provides the instructions for making cannabinoid components. Finally, the process is conducted on a large scale, which results in the production of materials that can be further processed into purified cannabinoids.
Piauhy Labs intends to obtain a Certification of Compliance with Good Laboratory Practices, in accordance with the OECD principles, for the pharmaceutical area, so that the results obtained from its research are properly used for the granting of licenses or for the registration of pharmaceutical products, including medicines for human use and similar products.
As a result of the intended research processes, Piauhy Labs wants to create patents and originate intellectual property, with the ultimate goal of producing innovative medicines, medicines that improve the quality of life of patients suffering from various diseases, notably diseases related to the central nervous system.
Elmo Resende, Ph.D
Director of R&D
Piauhy Labs
References
Belcher, M. S.; Mahinthakumar, J. and Keasling, J. D. New frontiers: harnessing pivotal advances in microbial engineering for the biosynthesis of plant-derived terpenoids. Current Opinion in Biotechnology 65, 88–93, 2020.
Braich, S.; Baillie, R. C.; Jewell, L. S.; Spangenberg, G. C. and Cogan, N. O. I. Generation of a Comprehensive Transcriptome Atlas and Transcriptome Dynamics in Medicinal Cannabis. Scientific Reports 9 (1), 16583, 2019.
Carsanba, E.; Pintado, M. and Oliveira, C. Fermentation Strategies for Production of Pharmaceutical Terpenoids in Engineered Yeast. Pharmaceuticals (Basel) 14 (4), 295, 2021.
Degenhardt, F.; Stehle, F. and Kayser, O. Handbook of Cannabis and Related Pathologies. Biology, Pharmacology, Diagnosis, and Treatment. Chapter 2 - The Biosynthesis of Cannabinoids 13-23, 2017.
Dusséaux, S.; Wajn, W. T.; Liu, Y.; Ignea, C. and Kampranis, S. C. Transforming yeast peroxisomes into microfactories for the efficient production of high-value isoprenoids. Proc. Natl. Acad. Sci. USA Dec 15, 117 (50), 31789-31799, 2020.
Gülck, T.; Booth, J. K.; Carvalho, Â.; Khakimov, B.; Crocoll, C.; Motawia, M. S.; Møller, B. L.; Bohlmann, J. and Gallage, N. J. Synthetic Biology of Cannabinoids and Cannabinoid Glucosides in Nicotiana benthamiana and Saccharomyces cerevisiae. J. Nat. Prod. 23, 83 (10), 2877-2893, 2020.
Rea, K. A.; Casaretto, J. A.; Al-Abdul-Wahid, M. S.; Sukumaran, A.; Geddes-McAlister, J.; Rothstein, S. J. and Akhtar, T. A. Biosynthesis of cannflavins A and B from Cannabis sativa L. Phytochemistry 164, 162–171, 2019.
Smith, M. "Biosynthesis of Cannabinoids in S. cerevisiae". Thinking Matters Symposium. 82. 2020.
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Biotransformations consist of chemical conversions of a substance (starting material) into a product, using whole cells of plants, microorganisms and animals, containing the necessary enzyme(s), or through enzymes isolated.
