proteinase A. Overall, a much higher specific activity was obtained inP. pastoris.
Whole cell bioconversion of CBGA in the recombinantP. pastorisproduced 0.36 g
THCA/L that is equivalent to 10.5 g of cell dry mass before the THCA synthase
activity was lost. These studies suggest an efficient system can be designed for
THCA production.
Besides recombinant microorganisms expressing THCA synthase originally
found inC. sativa, THCA production can also be accomplished in transgenic
tobacco (Nicotiana tabacum) hairy roots expressing the gene (Sirikantaramas et al.
2004 ). However, a lower yield of THCA was obtained upon CBGA feeding, rep-
resenting only 8.2% bioconversion from CBGA. This could be due to the toxicity of
both the substrate and product to tobacco hairy roots, as mentioned earlier.
Cannabinoid toxicity can also be observed inCatharanthus roseuscells in sus-
pension culture treated with THC (Akhtar et al. 2015 ), resulting in reduced dry cell
weight. Interestingly, glycosylated and hydroxylated derivatives of THC were
detected in the cell suspension. Since glycosylation is a well-known mechanism for
the detoxification of this toxic product (Sirikantaramas et al. 2014 ), it highly likely
thatC. roseuspossesses a gene encoding glycosyltransferase for THC detoxifica-
tion. Thisfinding also provides a route for the novel biotechnological production of
new cannabinoids that might exhibit interesting biological activities.
The frontier of synthetic biology has been successfully demonstrated to be
essential in the semi-synthetic production inS. cerevisiaeof the antimalarial arte-
misinin (Paddon et al. 2013 ) and opioid biosynthesis (Galanie et al. 2015 ).
Intriguingly, opioid production requires more than 20 enzymes from plants,
mammals, bacteria, and yeast itself. Recently, cyanobacteria have received much
attention as a host for production because they grow photoautotrophically. Several
codon-optimized plant genes encoding biosynthetic enzymes have been trans-
formed and the corresponding enzymes were successfully produced in
Synechocystis(Lindberg et al. 2010 ; Tantong et al. 2016 ). According to the com-
plete cannabinoid biosynthetic pathway starting from hexanoyl-CoA and
malonyl-CoA, either THCA or CBDA can be produced from just four different
enzymes (Figs.8.2 and8.10). Taken together, these results strongly suggest it
would be possible to develop a novel host for cannabinoid production in the near
future.
8.3.3 Production of Unnatural Cannabinoids
by Reengineering Cannabinoid Biosynthetic Genes
Although the reengineering of biosynthetic pathways has never been reported in
C. sativa, it has been successfully performed inC. roseus, allowing for the pro-
duction of unnatural monoterpene indole alkaloids. Runguphan and O’Conner
( 2009 ) redesigned the structure of strictosidine synthase, which is the key enzyme
that catalyzes the formation of strictosidine from secologanin and tryptamine. They
200 S. Sirikantaramas and F. Taura