Biosynthesis in the Plant

Two routes converge

Cannabinoid biosynthesis is a convergence, not a linear chain. One branch is a polyketide route that builds the aromatic resorcinol from fatty-acid-derived units. The other is the plastidial MEP route that builds the isoprenoid partner. Neither is cannabis-specific; both are general plant metabolism. What is cannabis-specific is the prenyltransferase that joins them and the three oxidocyclases that act afterwards. That is worth stating because it reframes what the plant is doing: it is not running an exotic pathway, it is running two ordinary ones into an unusual junction.

• Hexanoyl-CoA + 3 malonyl-CoA --[tetraketide synthase, a type III polyketide synthase]--> a linear tetraketide intermediate
• tetraketide intermediate --[olivetolic acid cyclase, OAC]--> olivetolic acid (the C5 resorcinolic acid)
• pyruvate and glyceraldehyde-3-phosphate --[plastidial MEP route]--> isopentenyl and dimethylallyl pyrophosphate --> geranyl pyrophosphate (GPP)
• olivetolic acid + GPP --[aromatic prenyltransferase, CBGA synthase]--> cannabigerolic acid (CBGA)
• CBGA --[THCA synthase]--> Δ9-tetrahydrocannabinolic acid (THCA)
• CBGA --[CBDA synthase]--> cannabidiolic acid (CBDA)
• CBGA --[CBCA synthase]--> cannabichromenic acid (CBCA)
• THCA, CBDA, CBCA --[non-enzymatic decarboxylation on heating, drying and ageing]--> THC, CBD, CBC

Sources: Gagne SJ 2012 · Sirikantaramas S 2004 · Radwan MM 2021 · Hanuš LO 2016

Why the 2012 olivetolic acid cyclase paper mattered

For years the polyketide arm had a hole in it. The type III polyketide synthase from cannabis trichomes had been cloned, but on its own it did not give olivetolic acid — it gave off-pathway products, principally a pyrone from a decarboxylative cyclisation, because the enzyme cyclises the tetraketide the wrong way and loses the carboxyl group that the rest of the pathway needs. Gagne and colleagues showed that a second protein, olivetolic acid cyclase, is required, and that it performs an intramolecular aldol condensation with retention of the carboxylate. That is the step that keeps the acid an acid. The paper was significant for three reasons: it completed the pathway, it identified a polyketide cyclase of a type that had not previously been described in plants, and it explained why earlier attempts to reconstruct cannabinoid biosynthesis outside the plant had stalled. Every subsequent heterologous reconstruction depends on it.

Sources: Gagne SJ 2012 · Luo X 2019

The three oxidocyclases and what they actually do

THCA synthase, CBDA synthase and CBCA synthase all take the same substrate, CBGA, and differ in how they close the geranyl chain onto the ring system. All three are FAD-dependent oxidases of the berberine-bridge-enzyme-like family: they carry a covalently bound flavin cofactor, they require molecular oxygen, and they produce hydrogen peroxide as a by-product. THCA synthase closes the pyran ring to give the tricyclic acid; CBDA synthase performs the corresponding oxidative cyclisation but leaves the ring system open, giving the bicyclic acid; CBCA synthase closes the chromene. The cloning and heterologous expression of THCA synthase in 2004 was, in the authors’ own framing, the identification of the gene controlling marijuana psychoactivity, and it established the enzymology for the family. One practical detail matters for anyone interpreting trichome chemistry: these enzymes are secreted and operate in the extracellular storage cavity of the glandular trichome, outside the cell that made them, which is unusual and which is part of why the plant tolerates producing a cytotoxic product.

Sources: Sirikantaramas S 2004 · Radwan MM 2021 · Hanuš LO 2016

Chemotype is genetics: the B locus

Which of the three synthases dominates is not an environmental outcome, it is inherited. De Meijer and colleagues crossed inbred pure-CBD and pure-THC lines, found that all the F1 plants had a mixed chemotype, and found that the F2 generation segregated pure-CBD, mixed and pure-THC in a 1:2:1 ratio. That is the signature of a single locus with two codominant alleles. The model they proposed — a B locus carrying B_D and B_T, where the heterozygote expresses both synthases and produces both acids — has held up as the working genetic account of chemotype. Five chemotypes are conventionally recognised: THC-predominant, mixed, CBD-predominant, a CBG-predominant type in which the routing step is impaired so the precursor accumulates, and a near-cannabinoid-free type. The consequence for the hemp industry is direct. Hemp versus marijuana is a genotype before it is a threshold: a B_D/B_D plant does not produce a THC-predominant profile under any growing conditions, and a B_T-carrying plant cannot be made compliant by agronomy. Threshold-based regulation of a genetically determined trait is testing for the consequence rather than the cause.

Sources: de Meijer EPM 2003 · Radwan MM 2021

The homologous series comes from the starter unit contested

The whole side-chain series is generated at the very first step, by which short acyl-CoA the polyketide synthase picks up. Hexanoyl-CoA, the abundant starter, gives olivetolic acid and hence the pentyl (C5) series. Butanoyl-CoA gives divarinic acid instead, and hence CBGVA and the entire propyl (C3) series in parallel — a complete second copy of the family. Shorter and longer acyl-CoA starters account, at least in principle, for the C1, C4, C6, C7 and C8 homologues found at trace and ultra-trace level. This is why the homologues are naturally occurring rather than exotic: the plant is not making them deliberately, it is exhibiting the substrate promiscuity of an early enzyme, and the abundance of each homologue tracks the availability of its starter. It is also the reason the pathway predicts compounds that have not yet been detected, which is the argument the research-frontier page builds on.

Contested — caveat. The acyl-CoA starter explanation is well established for the C5 and C3 series. For the C4, C6, C7 and C8 homologues it is a reasonable and widely accepted inference from the pathway rather than a demonstrated enzymology: the corresponding starter units and their incorporation have not been characterised enzymatically to the same standard.

Sources: Citti C 2019 · Linciano P 2020 · Linciano P 2020 · Hanuš LO 2016 · Gagne SJ 2012

The acids are the plant products; the neutral cannabinoids are mostly artefacts of what happens next

A living, unharvested cannabis plant contains very little Δ9-THC and a great deal of Δ9-THCA-A. The same holds for CBD and CBDA. The neutral cannabinoids that dominate the commercial and pharmacological literature are produced after the plant stops being alive, by decarboxylation during drying, curing, storage, extraction, heating and combustion. Two things follow. First, the pharmacology of cannabis as a plant and the pharmacology of cannabis as a product are not the same subject, and the acids deserve study in their own right rather than as precursors. Second, almost all of the receptor work in the literature was done on the neutral forms, so the acidic cannabinoids are systematically under-characterised relative to their actual abundance in the source material.

Sources: Wang M 2016 · Filer CN 2022 · Nadal X 2017 · Bolognini D 2013

Heterologous production exists, and this page does not describe how

Complete cannabinoid biosynthesis has been reconstructed outside the plant. Luo and colleagues reported in 2019 the complete biosynthesis of CBGA, THCA, CBDA and the corresponding varinic acids in engineered Saccharomyces cerevisiae, together with unnatural analogues obtained by feeding alternative starter units — which is the biosynthetic expression of the same starter-unit logic described above. Engineered microbial and cell-free routes are an active area, and they are relevant to this shelf for what they imply about the future supply of reference standards for rare homologues, which is a genuine analytical bottleneck. No strain construction, pathway engineering, fermentation or recovery detail appears here or anywhere in this shelf; the citation is given so that the primary literature can be consulted directly.

Sources: Luo X 2019 · Gagne SJ 2012