THCA synthase
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THCA synthase (tetrahydrocannabinolic acid synthase, THCAS) is the enzyme that turns cannabigerolic acid (CBGA) into tetrahydrocannabinolic acid (THCA), the compound the living plant accumulates and from which THC forms when the acid loses carbon dioxide.[1] It is one of three cannabinoid oxidocyclases known from cannabis, alongside CBDA synthase and CBCA synthase, and all three take the same precursor.[2] Which of these enzymes a plant has in working form settles its chemotype, the proportions in which it makes THC and cannabidiol.[3]
The gene was cloned in 2004, the first of the cannabinoid pathway to be isolated.[1] Plants of the drug crop carry a working copy, which the fibre and grain varieties of hemp lack, and the sequence has been used since the mid-2000s to tell the two kinds of plant apart before they flower.[4][5]
The reaction
THCA synthase closes a ring in the geranyl part of CBGA, an oxidative cyclisation. It carries a flavin adenine dinucleotide (FAD) cofactor, uses molecular oxygen and releases hydrogen peroxide, so it is a flavin-dependent oxidase and not an oxygenase or a peroxidase.[1] In the mechanism proposed from the crystal structure, a hydride passes from carbon 3 of CBGA to nitrogen 5 of the flavin while a proton is taken from the hydroxyl group of the substrate.[6]
Recombinant THCA synthase supplied with CBGA yields cannabidiolic acid (CBDA) and cannabichromenic acid (CBCA) besides THCA, in small amounts.[7] Measured at pH 5 and 30 °C, the enzyme was the most active and the most selective of the cannabinoid oxidocyclases tested, giving 95 per cent THCA and 5 per cent CBCA, where CBDA synthase gave 89 per cent CBDA and had an eighth of the activity.[8]
THCA itself is not psychotropic. It loses carbon dioxide without an enzyme as the harvested plant is stored and when it is heated or smoked, and the product of that loss is THC.[1]
Protein and structure
The gene encodes a polypeptide of 545 amino acids, of which the first 28 form a signal peptide; the mature protein of 517 residues has a predicted mass of 58,597 daltons. Its sequence resembles that of the berberine bridge enzyme of Eschscholzia californica, an enzyme of alkaloid synthesis.[1]
The structure was solved in 2012 at 2.75 ångström resolution. The protein has two domains with the flavin held between them, bound covalently to histidine 114 and cysteine 176. Changing histidine 114 to alanine leaves an enzyme with neither the light absorbance of a flavoprotein nor any activity.[1][6] Mutation of the active site showed that catalysis does not run through the glutamate 442 that acts as the catalytic base in the berberine bridge enzyme, but through the hydroxyl group of tyrosine 484; mutations at histidine 292 and tyrosine 417 lowered activity without abolishing it, which suits a part in holding the substrate rather than in catalysis.[6]
Structures of all three cannabinoid synthases, each with its flavin, were published in 2026, among them THCA synthase at 2.33 ångström. THCA synthase shares 93 per cent of its sequence with CBCA synthase and 84 per cent with CBDA synthase, and the three fold alike. Tyrosine 484, tryptophan 444, tyrosine 190 and histidine 292 are conserved in all three, while the substitutions that distinguish CBDA synthase alter the shape of its active site and the tunnel leading to it.[9]
Where it works in the plant
The transcript of the gene was found in the secretory cells of the glandular trichomes and nowhere else in the plant, activity was measured in the contents of the trichome storage cavity, and a fusion of the protein to green fluorescent protein lit up the trichome head in transgenic tobacco. The secretory cells therefore export the enzyme itself along with the metabolites, and the reaction runs outside them.[10]
THCA and CBCA applied to leaf tissue or to cultured cells of cannabis opened the permeability pores of the mitochondria, which swelled and lost their membrane potential, and the cells died.[11]
The gene and its forms
The gene has no intron and was cloned from rapidly expanding leaves of a Mexican plant. Tobacco hairy roots carrying the cloned sequence made THCA when they were fed CBGA, which established what the gene encodes.[1] Full-length coding sequences are found in plants of the drug crop, and sequence surveys group them into two main types and more than a dozen finer ones. Among them are sequences from a hashish landrace of Afghanistan, from Korean and Pakistani material, and from a plant of unclear status collected at Jilong in Tibet that carries six substitutions of its own.[2]
Thirteen strains sequenced in 2006 fell into six of the drug crop and seven of the fibre crop, each group with its own variant of the gene and 37 substitutions between the two deduced proteins. A PCR marker designed on the drug-crop variant did not amplify in the fibre-crop plants.[4] Sequences from plants of several chemotypes differed by single-nucleotide changes that track the cannabinoids the inflorescence ends up with.[12] Four of the changes separating fibre-crop from drug-crop plants in a panel of 21 varieties are expected to lower the enzyme's activity.[13]
A non-psychotropic Malawian plant carries the gene and accumulates CBGA in place of THCA, which has been put down to a single amino-acid change.[2] In two crossing populations, dominance of CBGA went with the homozygous state of a sequence carrying a guanine-to-adenine change at base 1,064, which puts asparagine in place of serine at position 355, and the trait was inherited as a single recessive.[14] Serine 355 is one of the residues by which the enzyme's activity was later traced back through its ancestors.[8]
The "inactive THCA synthase" amplified from European fibre varieties in the 2000s proved to be CBCA synthase, and the reports of several copies per plant are attributed to primers that picked up these neighbouring genes.[5][2]
Position in the genome
A map of the genome made from a cross between the drug strain Purple Kush and the hemp variety Finola found one copy of the gene in Purple Kush and none in Finola, which instead carries CBDA synthase. The two genes sit at separate, closely linked loci, each inside a stretch of more than 250 kilobases rich in retrotransposons that has almost nothing in common with its counterpart in the other parent, and both stretches lie within about 40 megabases of repetitive DNA where crossing over is rare. Purple Kush also carries a disabled copy of CBDA synthase, broken by an inserted gypsy element.[5]
In the genome of the cannabidiol-rich cultivar CBDRx the synthase genes form tandem arrays inside long terminal repeat retrotransposons on chromosome 7. That cultivar is mostly of drug-crop ancestry, carries CBDA synthase introgressed from hemp and has no complete copy of THCA synthase.[15]
Across 193 genomes assembled for a pangenome, no haploid genome held more than one full-length copy of THCA synthase or of CBDA synthase. The full-length genes sat in similar arrays of transposable-element cassettes, most of the other cassettes holding pseudogenes, generally within a region of about 1.5 megabases on chromosome 7. The synthase genes themselves carried very little variation although the region around them is among the most variable in the species, which the authors read as a sign of strong selection on these loci.[16]
Where the enzyme came from
The cannabinoid oxidocyclases belong to the berberine bridge enzyme-like family, within a group of that family which expanded separately in the mulberry and hemp families. In the hemp family the expansion produced a clade found in cannabis and not in its relatives, so these genes arose in the cannabis lineage rather than in an older duplication shared with hop.[2]
Three ancestral enzymes were reconstructed from 77 sequences of cannabis, hop and related plants and made in yeast. The oldest active one worked on CBGA and was unselective, giving 60 per cent THCA, 30 per cent CBDA and 10 per cent CBCA, where its descendants are specialists. Duplication followed by divergence is what narrowed that broad activity into the present THCA and CBDA synthases, and swapping single residues between ancestral and living enzymes shifted which cannabinoid they made.[8]
What it does for the plant
In a segregating population grown in the field, leaf damage by chewing insects fell as the cannabinoid concentration of the leaf rose, and caterpillars of Trichoplusia ni ate less and grew less on leaves richer in cannabinoids and on artificial diet to which cannabinoids had been added.[17] Li and colleagues reported in 2025 that the gene bears signs of positive selection among 17 accessions they tested under ultraviolet-B light, and proposed that THCA shields the plant from that radiation.[18]
Making the enzyme outside the plant
The enzyme has been produced in insect cells, in the yeast Komagataella phaffii and in tobacco.[1][7] A yeast strain in which three helper proteins were overexpressed held twenty times the activity of the strain it was bred from, and its cells converted CBGA into 3.05 grams of THCA per litre in eight hours at 37 °C.[19] Removing two of the enzyme's sugar attachment sites doubled its activity, and adding a disulfide bridge raised the temperature at which it worked best from 52 to 57 °C.[7] A strain of Saccharomyces cerevisiae carrying the whole pathway, the synthase included, made THCA from galactose alone.[20]
See also
References
- ↑ 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 Sirikantaramas, S.; Morimoto, S.; Shoyama, Y.; Ishikawa, Y.; Wada, Y.; Shoyama, Y.; Taura, F. (2004). "The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Δ1-tetrahydrocannabinolic acid synthase from Cannabis sativa L." Journal of Biological Chemistry. 279 (38): 39767–39774. doi:10.1074/jbc.M403693200.
- ↑ 2.0 2.1 2.2 2.3 2.4 van Velzen, R.; Schranz, M.E. (2021). "Origin and evolution of the cannabinoid oxidocyclase gene family". Genome Biology and Evolution. 13 (8) evab130. doi:10.1093/gbe/evab130.
- ↑ Wenger, J.P.; Dabney, C.J.; ElSohly, M.A.; Chandra, S.; Radwan, M.M.; Majumdar, C.G.; Weiblen, G.D. (2020). "Validating a predictive model of cannabinoid inheritance with feral, clinical, and industrial Cannabis sativa". American Journal of Botany. 107 (10): 1423–1432. doi:10.1002/ajb2.1550. PMC 7702092.
- ↑ 4.0 4.1 Kojoma, M.; Seki, H.; Yoshida, S.; Muranaka, T. (2006). "DNA polymorphisms in the tetrahydrocannabinolic acid (THCA) synthase gene in "drug-type" and "fiber-type" Cannabis sativa L.". Forensic Science International. 159 (2–3): 132–140. doi:10.1016/j.forsciint.2005.07.005.
- ↑ 5.0 5.1 5.2 Laverty, K.U.; Stout, J.M.; Sullivan, M.J.; Shah, H.; Gill, N.; Holbrook, L.; Deikus, G.; Sebra, R.; Hughes, T.R.; Page, J.E.; van Bakel, H. (2019). "A physical and genetic map of Cannabis sativa identifies extensive rearrangements at the THC/CBD acid synthase loci". Genome Research. 29 (1): 146–156. doi:10.1101/gr.242594.118. PMC 6314170.
- ↑ 6.0 6.1 6.2 Shoyama, Y.; Tamada, T.; Kurihara, K.; Takeuchi, A.; Taura, F.; Arai, S.; Blaber, M.; Shoyama, Y.; Morimoto, S.; Kuroki, R. (2012). "Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa". Journal of Molecular Biology. 423 (1): 96–105. doi:10.1016/j.jmb.2012.06.030.
- ↑ 7.0 7.1 7.2 Zirpel, B.; Kayser, O.; Stehle, F. (2018). "Elucidation of structure-function relationship of THCA and CBDA synthase from Cannabis sativa L.". Journal of Biotechnology. 284: 17–26. doi:10.1016/j.jbiotec.2018.07.031.
- ↑ 8.0 8.1 8.2 Villard, C.; Baser, I.; van de Peppel, A.C.; Cankar, K.; Schranz, M.E.; van Velzen, R. (2025). "Resurrected ancestral cannabis enzymes unveil the origin and functional evolution of cannabinoid synthases". Plant Biotechnology Journal. 24 (4): 2685–2697. doi:10.1111/pbi.70475.
- ↑ Domenech, J.; King, A.; Byrne, E.; Cartwright, J.; Grogan, G. (2026). "X-ray crystal structures of the cannabinoid synthases CBCAS, CBDAS and THCAS". Current Research in Structural Biology. 12 100197. doi:10.1016/j.crstbi.2026.100197.
- ↑ Sirikantaramas, S.; Taura, F.; Tanaka, Y.; Ishikawa, Y.; Morimoto, S.; Shoyama, Y. (2005). "Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavity of the glandular trichomes". Plant and Cell Physiology. 46 (9): 1578–1582. doi:10.1093/pcp/pci166.
- ↑ Morimoto, S.; Tanaka, Y.; Sasaki, K.; Tanaka, H.; Fukamizu, T.; Shoyama, Y.; Shoyama, Y.; Taura, F. (2007). "Identification and characterization of cannabinoids that induce cell death through mitochondrial permeability transition in Cannabis leaf cells". Journal of Biological Chemistry. 282 (28): 20739–20751. doi:10.1074/jbc.M700133200.
- ↑ Onofri, C.; de Meijer, E.P.M.; Mandolino, G. (2015). "Sequence heterogeneity of cannabidiolic- and tetrahydrocannabinolic acid-synthase in Cannabis sativa L. and its relationship with chemical phenotype". Phytochemistry. 116: 57–68. doi:10.1016/j.phytochem.2015.03.006.
- ↑ Cascini, F.; Farcomeni, A.; Migliorini, D.; Baldassarri, L.; Boschi, I.; et al. (2019). "Highly predictive genetic markers distinguish drug-type from fiber-type Cannabis sativa L." Plants. 8 (11) 496. doi:10.3390/plants8110496.
- ↑ Garfinkel, A.R.; Otten, M.; Crawford, S. (2021). "SNP in potentially defunct tetrahydrocannabinolic acid synthase is a marker for cannabigerolic acid dominance in Cannabis sativa L." Genes. 12 (2) 228. doi:10.3390/genes12020228.
- ↑ Grassa, C.J.; Weiblen, G.D.; Wenger, J.P.; Dabney, C.; et al. (2021). "A new Cannabis genome assembly associates elevated cannabidiol (CBD) with hemp introgressed into marijuana". New Phytologist. 230 (4): 1665–1679. doi:10.1111/nph.17243. PMC 8248131.
- ↑ Lynch, R.C.; Padgitt-Cobb, L.K.; Garfinkel, A.R.; Knaus, B.J.; Hartwick, N.T.; et al. (2025). "Domesticated cannabinoid synthases amid a wild mosaic cannabis pangenome". Nature. 643 (8073): 1001–1010. doi:10.1038/s41586-025-09065-0. PMC 12286863.
- ↑ Stack, G.M.; Snyder, S.I.; Toth, J.A.; Quade, M.A.; Crawford, J.L.; et al. (2023). "Cannabinoids function in defense against chewing herbivores in Cannabis sativa L." Horticulture Research. 10 (11) uhad207. doi:10.1093/hr/uhad207.
- ↑ Li, M.; Ouyang, Y.; Cai, S.; Zhang, Z.; Wu, Q.; et al. (2025). "A new UV-B protectant in plant: insights into THCAS and THCA's role in UV-B tolerance". Plant, Cell & Environment. 48 (10): 7729–7742. doi:10.1111/pce.70057.
- ↑ Zirpel, B.; Degenhardt, F.; Zammarelli, C.; Wibberg, D.; Kalinowski, J.; et al. (2018). "Optimization of Δ9-tetrahydrocannabinolic acid synthase production in Komagataella phaffii via post-translational bottleneck identification". Journal of Biotechnology. 272–273: 40–47. doi:10.1016/j.jbiotec.2018.03.008.
- ↑ Luo, X.; Reiter, M.A.; d'Espaux, L.; Wong, J.; Denby, C.M.; et al. (2019). "Complete biosynthesis of cannabinoids and their unnatural analogues in yeast". Nature. 567 (7746): 123–126. doi:10.1038/s41586-019-0978-9. PMC 6830570.