94NISR RESEARCH GRANT
2004 Young Investigator Research Grant

Production of Tetrahydrocannabinol by Transgenic Cultured
Cells
Futoshi TAURA
Graduate School of Pharmaceutical Sciences, Kyushu University
Research aims
It is known that tetrahydrocannabinol (THC), a major
pharmacologically active constituent of marijuana, is generated in its acidic form, tetrahydrocannabinolic acid(THCA), in the fresh tissues of Cannabis sativa. I have previously demonstrated that THCA is biosynthesized by anovel enzyme, THCA synthase, which catalyzes stereoselective cyclization of geranyl group of cannabigerolic acid(CBGA) to form THCA (Fig. 1). Since CBGA is easy to besynthesized from commercially available chemicals, andsince THCA is easy to be decarboxylated by heating,
THCA synthase would open the way to the novel biotechnological production system of THC. In this study, I attempted to establish an efficient expression system ofTHCA synthase, which is available for the biotechnologicalproduction of THC.
Methods
Expression of THCA synthase in a yeast culture—The
entire coding sequence of THCA synthase was subcloned
into the expression vector pPICZ-B (Invitrogen) containingalcohol oxidase promoter. The resulting vector was digested
with SacI, and transformed into a yeast Pichia pastoris
SMD1168 (Invitrogen). The transformed colonies were selected on minimal plates containing zeocin, and subcultured
on the same plates. The transformed Pichia harboring
THCA synthase gene was inoculated in liquid BMGY complex medium containing glycerol, and cultured for 2 days at30°C with gentle shaking. Then, the cultured cells were col-lected by centrifugation, and resuspended in BMMY complex medium containing methanol to induce the expressionof the recombinant THCA synthase. The THCA synthaseactivities in the culture medium and cell extract were measured by HPLC.
Expression of the recombinant THCA synthase in tobacco hairy roots—Full-length THCA synthase cDNA wassubcloned into the plant expression vector pBI121 (Clontech). The resulting construct was introduced into Agrobacterium rhizogenes strain 15834 by tri-parental mating. Thetobacco (Nicotiana tabacum cv. Xanthi) stems were infected with A. rhizogenes using needles. Hairy roots generated from the infected sites were cultured on solidified B5
media containing cefotaxime and kanamycin. For the bioconversion of CBGA into THCA, 2 cm tips of the root
expressing THCA synthase were inoculated in 30 ml of liquid B5 medium in 100-ml flasks and pre-cultured for 2weeks at 25°C with gentle shaking (90 rpm). Then, 1 mg ofCBGA was added to the medium, and the hairy roots were
further cultured under the same conditions to produce
THCA.
Expression of the recombinant THCA synthase in tobacco BY-2 culture—pBI121 expression vector harboring
THCA synthase was introduced into Agrobacterium tumefaciens strain LBA4404 by tri-parental mating. The tobacco
BY-2 cells were transformed by infection with Agrobacterium. Transformed cells were selected on solidified BY-2
media containing cefotaxime and kanamycin. For the bioconversion of CBGA into THCA, transformed BY-2 callus
expressing THCA synthase were inoculated in 30 ml of liquid BY-2 medium in 100-ml flasks and pre-cultured for 1weeks at 25°C with gentle shaking (90 rpm). 1 mg ofCBGA was added to the culture flask or the cell-free culture supernatant, and incubated under the same condition toproduce THCA.
ResultsFig. 1. Scheme of THC formation. THCA synthase catalyzes oxidative
cyclization of the monoterpene moiety of CBGA to form THCA. THC isderived from THCA by non-enzymatic decarboxylation.

Heterologous expression of THCA synthase in yeast—We
first tried a yeast expression of THCA synthase because theyeast system is easy to construct. Interestingly, enzyme assays demonstrated that the THCA synthase activity in themedium (32 picokatal/l) was much higher that that (1.8 picokatal/l) of the cell extract, indicating that most of the recombinant enzyme was secreted. The secretion level of therecombinant THCA synthase was 9-fold improved (0.30nanokatal/l) by adding some supplements such as casamino

95Fig. 2. Development of THCA-producing tobacco hairy roots. A, induction of hairy roots from tobacco stem infected with Agrobacterium rhizogenes harboring THCA synthase cDNA. B, Production of THCA in suspension cultures of transgenic tobacco hairy roots expressing recombinant
THCA synthase. The roots were inoculated in 30 ml of liquid Gamborg B5
medium and pre-cultured for two weeks. Then, 1 mg of CBGA was added
to the culture, and the accumulation of THCA in the aliquot of hairy roots
and medium was measured by HPLC.

acid and proteinase inhibitors, which are known to stabilize
the secreted proteins in yeast cultures. The maximum expression level was estimated to be less than 0.2 mg/l culture
based on the specific enzyme activity of the purified native
THCA synthase (1.9 nanokatal/mg protein). The yeast expression demonstrated that THCA synthase cDNA actuallyencodes an active enzyme. However, the expression level
was unexpectedly low, and should be improved for furtherinvestigation.
Heterologous expression of THCA synthase in tobacco
hairy roots—For the expression in plants, THCA synthasegene was cloned into a pBI121 vector, and introduced into
the tobacco genome using Agrobacterium rhizogenes. The
transformants appeared as rapidly growing hairy roots fromtobacco stem infected with Agrobacterium because of theco-integration of bacterial root-forming genes (Fig. 2A). I
confirmed that the hairy roots possessed THCA synthaseactivity (1.2 picokatal/mg protein).
I next investigated whether transformed hairy root canproduce THCA. When the hairy roots were cultured in liquid B5 medium (30 ml) supplemented with 1 mg of CBGA,the maximum level of THCA (82 m g, 8.2% conversion fromCBGA) was produced 2 days after the addition of CBGA
(Fig. 2B). Although the hairy roots did not secrete THCA
synthase, about half of the THCA produced was found in
the culture medium, indicating CBGA uptake and THCA
release by tobacco cells. This is the first evidence thatTHCA synthase can produce THCA not only in Cannabis
sativa but also in transgenic plant. However, since the conversion rate was not improved by various modifications for
culture and reaction conditions, I suspected that the expression system is still insufficient for the cannabinoid production.
Production of THC by using the recombinant THCA synthase secreted from transgenic BY-2 cells—To improve theexpression level of the recombinant THCA synthase, I used
tobacco BY-2 cell as the third expression host because thiscell line grows more rapidly than any other plant cell or tissue cultures including hairy roots. Enzyme assays with cell

extract and culture medium from transformed BY-2 liquid
cultures demonstrated that most THCA synthase activity(95%) was secreted into the culture medium as described
for the yeast expression. The most active cell line afforded
a THCA synthase activity of 8.7 nanokatal/l culture
medium. This enzyme activity suggested the amount of theexpressed THCA synthase is more than 4 mg/l as the specific activity of the purified native THCA synthase is1.9 nanokatal/mg protein. Thus the expression level wasgreatly improved by using BY-2 cells as host.
For the production of THCA by the BY-2 culture, I firstattempted the bioconversion by feeding of CBGA directly
into the cell suspension culture. However, the conversion
rate was unexpectedly low (810%) in spite of the high expression of THCA synthase. I suspected that, in the culture,
metabolic enzymes other than THCA synthase degrade
CBGA, because the decrement of CBGA was much fasterthan THCA production. Therefore, I next removed the cells
from the culture by filtration through nylon screen, andadded CBGA into the supernatant. As a result, up to 45%
conversion into THCA was achieved after 6-hour incubation with the culture supernatant as enzyme. The enzymatically synthesized THCA was quantitatively decarboxylatedby heating at 120°C, and resulting THC was readily purified by passing a short silica gel column with benzene as amobile phase. Thus, in this study, I have established a novel
biotechnological production system of THC based on transformed tobacco BY-2 cell cultures.
Conclusion
In the present study, I have expressed the recombinant
THCA synthase in three different heterologous expression
systems, a yeast expression system and plant expression
systems using different hosts (tobacco hairy roots and BY-2cells). The yeast and tobacco hairy root systems produced
active THCA synthase, however, the expression level of theenzyme was not enough for effective THCA production. On
the other hand, the transformed BY-2 culture secreted alarge amount (4 mg/liter) of the enzyme. The supernatantobtained from the culture could efficiently convert CBGA
into THCA, which is later decarboxylized into THC. Therefore, I have established here a novel production system ofTHC based on the tobacco BY-2 cells expressing THCA
synthase.
References
1.

2.

S. Sirikantaramas, S. Morimoto, Y. Shoyama, Y. Ishikawa, Y. Wada,
Y. Shoyama and F. Taura “The gene controlling marijuana psychoactivity: Molecular cloning and heterologous expression of D1-tetrahydrocannabinolic acid synthase from Cannabis sativa L.” J. Biol.
Chem., 279, 39767–39774 (2004).
S. Sirikantaramas, F. Taura, Y. Tanaka, Y. Ishikawa, S. Morimoto andY. Shoyama “Tetrahydrocannabinolic acid synthase, the enzyme controlling marijuana psychoactivity, is secreted into the storage cavityof the glandular trichome” Plant Cell Physiol. (2005) in press.