Cannabinoid biosynthesis: How are CBD and THC produced?

One of the greatest pleasures of knowledge is to discover that it never ends. The topic we will discuss today in the blog is the synthesis or biosynthesis of cannabinoids. And why is the study of how cannabinoids are produced important? Well, for many things actually.

The exciting world of cannabinoid biosynthesis

Cannabinoid biosynthesis is an area of great research interest in the cannabis industry and medicine, as these compounds have a wide range of potential therapeutic properties.

Understanding the processes for producing cannabinoids can help improve the production of specific cannabinoids for use in medical treatments. But, also, understanding the complexity of this process allows us to better understand the arbitrariness of the THC limits allowed for cannabisThe number of children under the age of five, which in many countries is less than 1%, is totally ridiculous, as it goes against biology itself.

What is cannabinoid biosynthesis?

Cannabis plants produce chemical compounds known as phytocannabinoids through a biological process called cannabinoid biosynthesis. This process occurs in the trichomes of cannabis .

The biochemical pathway of cannabinoid synthesis.

The biochemical pathway by which the cannabinoids we know are produced is long and involves: non-cannabinoids, cannabinoids and enzymes.

Furthermore, as a curiosity, in the cannabis plant, the upstream steps of cannabinoid biosynthesis converge with the pathway of the terpenes .

It all starts with CBGA (cannabigerolic acid), which is the mother of the other cannabinoids, from which THCA (Δ-9-tetrahydrocannabinolic acid), CBDA (cannabidiolic acid) and CBCA (cannabichromenic acid) will be produced. And, although we don’t fully know the pathway of other cannabinoids, cannabis produces a hundred different cannabinoids!

As mentioned in a previous writing, the Cannabis sativa plant produces cannabinoid compounds in an acidic form, represented by an “A” at the end of each cannabinoid’s name (e.g. CBDA, THCA).

The marijuana plant produces cannabinoids in acid form, and these, when heated, change to their neutral form by a process called decarboxylation.

Upon heating, a chemical process called decarboxylation occurs, where these compounds change to their neutral form [1]. During this chemical process, the cannabinoids cannabinoids in acid form lose a carboxyl (COOH) group and form the CBD (cannabidiol) , THC (Δ-9-tetrahydrocannabinol) and CBC (cannabichromene).

This neutral form of cannabinoids is the one that interacts the most with our endocannabinoid system [2]. For this reason, to obtain the effect of cannabis we smoke, vape, infuse or cook the plant.

This decarboxylation process happens outside the plant, in other words, the plant produces the compounds in the acid form, and the neutral form occurs on the outside, which I refer to in the figure as a ‘non-enzymatic conversion’.

Enzymes of cannabinoid biosynthesis

What are enzymes?

The synthesis of cannabinoids within the plant occurs thanks to proteins called enzymes. To simplify, we could say that enzymes are a type of protein that performs a function, does something. In this case, the enzymes of cannabinoid synthesis act as an architect that modifies or transforms one molecule into another.

In reality, these changes occur in many ways: sometimes by trimming the chemical molecule, but sometimes by folding it, or by gluing a chemical group, and so on. In cannabis, cannabinoid biosynthesis begins with the enzyme CBGAS (cannabigerolic acid synthase), which produces the “mother cannabinoid”: CBGA.

Then, there are enzymes that are in charge of transforming CBGA into other cannabinoids: THC synthase, CBD synthase and CBCA synthase. So far it seems very schematic and simple, doesn’t it? Well, this is not always the case: as we shall see, these enzymes have a lot to say.

The mother cannabinoid: CBGA

The three enzymes THCA, CBDA and CBCA synthases act on CBGA, which is the precursor of these three cannabinoids. So, CBGA is the mother that gives birth to these cannabinoids. This is one of the reasons why I like CBGA, because as a mother, I identify with other mothers.

CBGA is the cannabinoid used by enzymes in the cannabis plant to produce the cannabinoids THCA, CBDA and CBCA and for this reason it is known as the mother cannabinoid.

In the figure I show the last two of the many steps that occur within the plant in the biosynthesis of cannabinoids:

Cannabinoid biosynthesis, metabolic pathway, cannabinoid synthesis
Figure: Biosynthesis of cannabinoids and enzymes involved. Final steps of the metabolic pathway by which the known cannabinoids THCA and CBDA are produced. Figure modified from references 3, 4, 5, and 6. Credit: Dr. Daniela Vergara.

The figure shows the last steps of the metabolic pathway by which the cannabinoids CBC, CBD and THC are produced. The enzyme CBGA synthase (CBGAS) converts geranyl diphosphate to cannabigerolic acid(CBGA). CBGA is the precursor molecule used by the enzymes THCA, CBDA, and CBCA synthases to produce the cannabinoids THCA, CBDA, and CBCA, respectively. Upon heating, these three compounds, as well as CBGA, decarboxylate to their neutral forms THC, CBD, CBC, and CBG. This decarboxylation step occurs outside the plant, which I call a ‘non-enzymatic conversion’.

The cannabinoid great-grandmother

Wondering what the ancestor of cannabinoids is? If CBGA is the mother cannabinoid, the great-grandmother of all cannabinoids is palmitic acid. Like the endocannabinoids phytocannabinoids are synthesized from fatty acids .

Promiscuous and neglected enzymes: Pure revelry!

As you can see in the figure, there are many different enzymes involved in cannabinoid synthesis. These proteins perform a task, in this case synthases, as they catalyze (cause, provoke) a reaction, which gives rise to the various cannabinoids.

The enzyme CBGAS (cannabigerolic acid synthase) converts olivetolic acid (OLA; derived from palmitic acid) and geranyl diphosphate into cannabigerolic acid. Chemical readers will tell me if I am translating these names properly from English to Spanish.

The cannabinoid oxidocyclases are the well-known THCA synthase, CBDA synthase and CBCA synthase, which take cannabigerolic acid and convert it to THCA, CBDA and CBCA, respectively.

In terms of chemical structure, CBDA has a cyclic structure of more, compared to the enzyme CBGA synthase. This structural difference translates into differences in their biological effects.

This terminology of ‘cannabinoid oxide cyclases’, which I love, I take from van Velzen and Schranz’s 2021 paper [7], which I commend to you.

As a curiosity, if we get biochemical, it is for this reason that van Velzen and Schranz call the THCA, CBDA, and CBCA synthases cannabinoid oxidocyclases: oxide because they remove a hydrogen from CBGA; and cyclases because THCA, CBDA, and CBCA cannabinoids all have one more ring.

Characteristics of cannabinoid synthesis enzymes

Now, although many of us were taught in undergrad that enzymes are very specific, as a key is to a lock, this is not the case with cannabinoid oxidocyclases (THCA, CBDA and CBCA synthases).

The enzymes involved in cannabinoid synthesis can produce up to eight different compounds in vitro, including the compound in the other [8]. And, apparently, also in the plant in vivo these enzymes are not very specific [8], although many studies are needed to confirm what, and in what amounts, these enzymes are producing in the plant.

That is: the enzyme that produces THCA (THCA synthase) can, in addition to producing THCA, produce CBCA, and CBDA. The same is true for CBDA synthase: it can also produce THCA and CBCA and so on, up to eight compounds.

This is called ‘sloppyness’, which I translate to sloppy enzymes. And according to my hypothesis, yet to be confirmed, the most neglected of all is CBCA synthase, but this is also another story for later.

The enzymes that produce cannabinoids are promiscuous and sloppy: they can produce up to eight different cannabinoids in vitro, including each other.

Moreover, as these three enzymes use CBGA as a precursor compound, and it is the same compound where all three act, they could also be categorized as promiscuous enzymes.

As you can see, our little enzymatic friends are out and about, promiscuous and careless, producing the compound of the one, or the other, or other compounds out there, a la topa tolondra (which, if you go to Cali Colombia, I recommend you to go dancing at that discotheque).

CBGA synthase: The intriguing genetic structure of the mother cannabinoid enzyme

Something that I found quite interesting is the genetic structure of the gene that codes for the CBGA synthase enzyme. This gene has many exons, and therefore also introns [6]. Exons are the part of the gene that has the information to produce proteins, while introns have no information.

Although both exons and introns are found in the DNA or genetic material of cannabis, forming part of the gene that produces CBGAS, when the enzyme or protein is produced, it only has the genetic information of the exons.

Characteristics of the CBGAS enzyme

What I find interesting about this genetic structure is that the introns are quite large, up to almost 11,000 nitrogen base pairs (letters; [6]), and, although introns of this size have been reported, they are not that common.

In addition, this gene has many introns, nine, ten, and even eleven [6].

So, it is a gene with quite a few pieces, which is also interesting because several types of proteins can be produced, which hopefully will be studied scientifically in the near future.

The gene encoding the CBGA synthase enzyme has an interesting structure with several exons and introns. Exons are the part of the gene that is translated into protein, and having several of these provides the possibility of forming different protein structures.

This is another reason why CBGA is my favorite cannabinoid, because, although scientific studies are lacking to confirm this hypothesis, suddenly, the gene can produce different protein structures, thus being versatile, moldable, and giving, as we mothers are.

How are the minor cannabinoids CBCA and CBGA produced?

Because many of the cannabinoids such as CBCA and CBGA are produced in the cannabis plant in small amounts, especially when compared to THCA and CBDA [9], they are referred to asminor cannabinoids (minor cannabinoids). Even so, these apparently have pharmacological properties and possible medicinal uses.

Synthesis of minor cannabinoids

There are many other minor cannabinoids, such as CBCA and CBGA, and THCVA and CBDVA.

These are produced in an alternate route using divarinolic acid instead of olivetolic acid, which we will discuss on another occasion.

Other cannabinoids, such as CBN, are produced by oxidation (without enzymes) from THC, so CBN could be categorized as a breakdown product.

Cannabinoid enzymes and their role in the cannabinoid profile of cannabis strains

Is it possible to select which cannabinoids we want a cannabis plant to produce?

For many years, and in a clandestine way, the methods used to select which cannabinoids we want a plant to produce were through artificial selection, that is, selecting plants with the desired characteristics, based on aroma, potency, vigorousness…

Marijuana growers, informally, knew about cannabinoid biosynthesis long before scientists described these chemical reactions.

Artificial selection has been used in many plants and other organisms. For example, in dogs, to obtain varieties, or different breeds (cultivars, if we apply it to cannabis), with desired characteristics.

But behind this ancient technique, there is cannabinoid chemistry. For example, THC-free marijuana has been achieved with crosses of high-CBD strains. Genetically, cannabis plants very high in CBD have the gene that produces the enzyme THCA synthase truncated or defectiveThe cannabinoid THC is not produced in the plant, or not as much (because, remember, they are neglected enzymes), and other cannabinoids, such as CBD, are accumulated.

In contrast, strains that are very high in THC, with very psychoactive effects, tend to have the gene for the CBDA synthase enzyme misfired.

This is one of the reasons why the production of cannabinoids in cannabis plants can vary significantly depending on genetics, although aspects of cultivation (lighting, irrigation, soil pH, temperature…) also play a role.

Cannabinoids on demand? Not so easy

One might think that, if we want strains that do not produce a type of cannabinoid, we could silence the genes that produce the particular synthase enzyme. But this is not the case: As we have seen, these enzymes are neglected, and all of them, in theory, are capable of producing THC, and in practice we see that, when there are plants that produce high CBD, they usually also have THC, even if in small quantities.

Ignorant Legislators

It is quite likely that those legislators who wrote the hemp and marijuana rules had no idea that these enzymes were on the rampage. Because had they known that cannabinoid enzymes were promiscuous and sloppy, perhaps they would have set the THC limit higher than 0.3%, more plausible with plant biology, or no limit at all.

The profound ignorance of the plant’s biochemistry and biology by legislators and regulators harms cannabis growers, producers, and breeders.

On a previous occasion I had told you about my deep anger and frustration due to these unjust and absurd regulations about the types of marijuana .

If they see these ignorant legends out there, tell them that there is an evolutionary biologist and cannabis researcher out there strongly criticizing their uninformed, unconscious and selfish decisions.

Referencias
  1. Hart, C.L., et al., Effects of acute smoked marijuana on complex cognitive performance. Neuropsychopharmacology, 2001. 25(5): p. 757-765.

Gertsch, J., et al., Beta-caryophyllene is a dietary cannabinoid. Proceedings of the National Academy of Sciences, 2008. 105(26): p. 9099-9104.

3. Page, J.E. and J.M. Stout, Cannabichromenic acid synthase from Cannabis sativa. 2017, Google Patents.

4. Vergara, D., et al., Gene copy number is associated with phytochemistry in Cannabis sativa. AoB PLANTS, 2019. 11(6): p. plz074.

5. Gülck, T. and B.L. Møller, Phytocannabinoids: origins and biosynthesis. Trends in plant science, 2020. 25(10): p. 985-1004.

6. Innes, P.A. and D. Vergara, Genomic description of critical upstream cannabinoid biosynthesis genes. bioRxiv, 2022: p. 2022.12. 15.520586.

7. van Velzen, R. and M.E. Schranz, Origin and evolution of the cannabinoid oxidocyclase gene family. Genome Biology and Evolution, 2021. 13(8): p. evab130.

8. Zirpel, B., O. Kayser, and F. Stehle, Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativa L. Journal of biotechnology, 2018. 284: p. 17-26.9. Smith, C.J., et al., The phytochemical diversity of commercial cannabis in the United States. PLoS one, 2022. 17(5): p. E0267498.

Information on cannabinoid synthesis (frequently asked questions)

What is cannabinoid synthesis?

Cannabinoid biosynthesis describes the chemical reactions that occur in the cannabis plant by which cannabinoids originate. The process of cannabinoid biosynthesis involves a series of enzymatic steps that convert precursor chemicals into active cannabinoids. The main precursor of cannabinoids is cannabigerolic acid (CBGA), which is converted into THC, CBC or CBD, through the action of specific enzymes.

Where does cannabinoid synthesis occur?

In cannabis plants, cannabinoid biosynthesis occurs in the glandular trichomes, which are specialized structures found on the surface of the plant’s leaves, stems and, above all, flowers. However, synthetic cannabinoids can also be produced.

Dra. Daniela Vergara
Investigadora y catedrática | Especialista en cultivos emergentes y consultora de cannabis

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