The evolutionary plasticity of cannabinoid synthases and their role in minor cannabinoids production

In Cannabis, more than 144 cannabinoids have been isolated and identified (Hanu? et al., 2016). While most research has focused on THC and CBD, many other minor cannabinoids have interesting pharmacological properties. Historically minor cannabinoids have been disregarded as degradation products because of their negligible quantities. Although this classic idea is still perpetuated, in recent years, evidence is starting to pile up, and an entirely new narrative behind minor cannabinoids and cannabinoid synthases (the enzymes synthesizing them) is revealing itself. Here I would like to take you through the mechanisms which give rise to the incredible number of cannabinoids and highlight how we can harness these natural processes to discover and create new medicine.

How chemical diversity arises in plants

Gene duplication is the primary genomic mechanism that drives new enzyme evolution (Kliebenstein, 2008). When a gene coding for an enzyme is duplicated, it results in a release of its functional constraints. In fact, while the original gene is still performing its job, the duplicated genes are free to accumulate random mutations without negatively affecting plant fitness (Zhang, 2003). Most of the time, mutations in the duplicated gene ultimately result in its degeneration into a nonfunctional gene. On the other hand, if the new function that has arisen confers an advantage, it can result in its neofunctionalization (Weng et al., 2012).

The newly duplicated and subsequently mutated enzymes often display lower catalytic efficiency but an increased acceptance of multiple (similar) substrates and/or synthesis of multiple products (Weng et al., 2012). The central feature of this evolutionary plasticity is that just a single or few amino acid mutations in the active site can lead to a different product profile (Xu et al., 2017). Therefore, these genetic processes appear to play a prominent role in the evolution of chemical diversity. Duplications can generate raw genetic material. Mutations can "open up" the enzyme to accept new related substrates and/or synthesize several new "by-products". While later on, natural selection can increase the enzyme affinity and efficiency toward producing the new beneficial compounds (Khersonsky et al., 2006).

Several examples suggest that in specialized metabolic pathways, natural selection favours the maintenance of a certain degree of enzymatic multifunctionality (Copley, 2017). First, synthesizing multiple ecologically beneficial products from a single enzyme is metabolically cost-effective. Secondly, retaining a structure which enables a certain degree of multifunctionality upon mutations ensures a fast and efficient way to evolve new gene functions.

The extraordinary catalytic capabilities of the Cannabinoid synthases

Cannabinoid synthase genes have evolved through repeated duplication events (Laverty et al., 2019). They originated and are part of the BBE-like gene family, notorious for their catalytic versatility and significant role in generating biochemical novelty (van Velzen & Schranz, 2021; Daniel et al., 2017). Interestingly, cannabinoid synthases have shown a remarkable ability to be promiscuous in substrate acceptance and product synthesis, highlighting their pivotal role in the rise of cannabinoid diversity. Indeed, a study by Zirpel et al. (2018) showed that when fed CBGA to recombinant purified CBDA synthase (CBDAS) and THCA synthase (THCAS), the enzymes produced their main product as well as minor quantities of CBDA, THCA, CBCA and three other non-identified cannabinoids. Additionally, Flores-Sanchez et al. (2010) suggested that different polyketide synthases may be responsible for synthesizing cannabinoid precursors with a different number of carbon atoms on the alkyl side chain. Typically, cannabinoids present 5 carbons on the alkyl side chain, but analogues with 1, 3,4,6 and 7 carbons can be naturally present in small quantities (de Meijer & Hammond, 2016; Citti et al., 2019; Linciano et al., 2020). It is supposed that cannabinoid synthases might be able to accept multiple substrates and produce these alkyl analogues in minor quantities in planta. Luo et al. (2019) provided strong evidence to support this theory by engineering the cannabinoid biosynthetic pathway in yeast. They observed that when fed different fatty acids in the pathway, several CBGA alkyl analogues were produced, which were subsequently converted by THCAS in the respective THCA alkyl analogues.

Although the common idea is that most minor cannabinoids are degradation products of THC and CBD, it seems that, instead, the synthases might be responsible for at least a good portion of them.

For example, considering the results of the studies of Zirpel et al. (2018) and Luo et al. (2019), a single enzyme able to produce six different products and accept at least six different precursors could potentially synthesize 36 different cannabinoids.

Even more surprising is the degree of variability in these genes. More than 200 different cannabinoids synthases variants have been deposited in the NCBI database, and recent genomic data revealed additional new cannabinoid-like genes (Grassa et al., 2021; McKernan et al., 2020; Vergara et al., 2019; van Velzen & Schranz, 2021).

Taura et al. (2007b) suggested that only a small number of amino acid residues would determine their product specificity, highlighting the possibility that specific enzyme variants could synthesize some minor products more efficiently. Produce different minor product combinations or, even more tempting, that specific variants are indeed a novel unidentified synthase (like in the case of CBCAS discovered by Laverty et al. in 2019).

Further studies with recombinant cannabinoids, cannabinoid-like and closely related BBE-like synthases testing multiple variants (obtained from plants or using directed evolution and/or rational design) could shed light on how in planta cannabinoids diversity is produced and how it has evolved.

Moreover, the production of minor cannabinoids via heterologous expression could increase the availability of rare or even novel cannabinoids, speeding up their research and application as medicine.

In the meantime, some of the outstanding questions which remain unanswered are:

1) Can cannabinoid synthase copies with slightly changed product formation explain minor cannabinoid diversity?

2) What are the products from cannabinoid synthases-like and closely related BBE-like enzymes found in the Cannabis genomes?

3) What is the biological activity of cannabinoid cocktails produced via heterologous expression?

As always, if you are interested in the studies, have a look at the references and if you find this article interesting, share it with your network!

References

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