toxic to the hairy root culture. EngineeringC. sativatissue cultures to overexpress
genes involved in cannabinoid biosynthesis could enhance the production level of
this compound, although it would, of course, be necessary to consider the toxicity
of cannabinoids produced in the culture.
It is important to explore how to reduce this toxicity to hosts involved in pro-
duction. Studies on compartmentalization of self-produced defense-related bioac-
tive compounds provide relevant information (Sirikantaramas et al. 2008 , 2014 ).
Absorbents such as polystyrene resin might be added to the culture medium to
absorb the toxic compound or stimulate biosynthesis (Saito et al. 2001 ; Cai et al.
2012 ). Andre et al. ( 2016 ) suggested an interesting approach to avoid toxicity in the
THCA production system using artificial compartmentalization, which has been
shown to function inArtemisia annuacell cultures for the production of artemisinin
(De Sansebastiano et al. 2015 ). The toxic cannabinoids produced can be trapped in
an artificial compartment similar to the storage cavity of the glandular trichome of
C. sativa.
A recent study on alterations in seed fatty acid composition ofC. sativausing an
approach involving a EST library and whole genome sequence mining, identified
desaturase genes required for the production of polyunsaturated fatty acids in seeds
(Bielecka et al. 2014 ). They screened an ethyl methane sulfonate (EMS)
mutagenized population using the TILLING method and found non-sense mutation
in the desaturase genes related to the function of the enzymes. This strategy could
also potentially be useful for manipulating cannabinoid production.
8.3.2 Cannabinoid Production in a Heterologous Host
Success cloning of both THCA synthase and CBDA synthase enabled the pro-
duction of THCA and CBDA in heterologous hosts upon feeding CBGA to the cells
maintained in a culture medium. CBGA is rather easy to chemically synthesize
compared to THCA and CBDA. In fact, insect (Spodoptera frugiperda) cells were
thefirst non-plant heterologous hosts to be used for the characterization of a
recombinant THCA synthase and CBDA synthase (Sirikantaramas et al. 2004 ;
Taura et al.2007a,b). Both recombinant enzymes had N-terminal signal peptides
and were secreted into culture medium, allowing for easy purification. Although the
activity of the recombinant THCA synthase was similar to that of the native
enzyme, the recombinant CBDA synthase showed relatively low levels of activity.
Secreted recombinant THCA synthase was also obtained fromPichia pastoris
(Taura et al.2007a,b). An improved production of THCA synthase inP. pastoris
has been shown in 2 L bioreactors, in which the purified enzyme was used in a
two-liquid phase setup leading to THCA production at a milligram scale (Lange
et al. 2015 ). This study provides a novel method for cannabinoid production in a
cell-free system. Interestingly, Zirpel et al. ( 2015 ) reported the production of an
intracellular THCA synthase in Saccharomyces cerevisiae and Pichia
(Komagataella) pastoris using a signal peptide from the vacuolar protease,
8 Cannabinoids: Biosynthesis and Biotechnological Applications 199