separate (Taura 2009 ; Bell 2016 ). Moreover, as Cannabis is governed by interna-
tional drug control conventions, most nations have policies in place to prohibit the
cultivation and use of Cannabis, despite its reportedly diverse benefits to society
(Bifulco and Pisanti 2015 ; Rehm and Fischer 2015 ; Spithoff et al. 2015 ). Thus,
government regulations restrict thefield cultivation of hemp and marijuana, placing
an additional limit on the supply of cannabinoids that can be extracted from
C. sativa. Furthermore, chemical synthesis of THC is made difficult by the high cost
of chiral precursor molecules and low yields (Zirpel et al. 2015 ). Minor cannabi-
noids are now attracting interest as potential medicines (Hill et al. 2012 ; see
Sect.16.4.3.3). As these compounds appear in trace amounts in Cannabis extracts,
alternative production methods are required to synthesize them in higher concen-
tration so they can be studied. Taken together, conventional methods are not
practical to supply enough pure cannabinoids to meet the demand (Zirpel et al.
2015 ). To move forward and navigate around these issues, research is being
directed toward metabolically engineering the cannabinoid pathway in other host
organisms.
16.4.3.1 Over-Expression of Tetrahydrocannabinolic Acid Synthase
in Tobacco Hairy Root Cultures
One of thefirst attempts toward synthesizing THCA in a heterologous host was to
develop an in vitro plant expression system for THCAS (Sirikantaramas et al.
2004 ). The CannabisTHCAS gene was cloned into an expression vector and
introduced intoA. rhizogenesfor transformation of tobacco. Biochemical analysis
demonstrated that tobacco hairy root cultures possessed THCAS activity. Unlike
the over-expression of THCAS in insect cells (see Sect.16.4.2.1) and tobacco
leaves (see Sect.16.4.1.1), tobacco hairy roots did not secrete the enzyme, sug-
gesting that different hosts or tissue types possess distinct sorting mechanisms for
the enzyme. Upon supplementing the hairy root culture medium with CBGA, the
precursor was taken up by hairy roots and converted to THCA (Fig.16.1). These
results provided direct evidence for a functional recombinant enzyme and
demonstrated that THCA, a valuable metabolite, can be synthesized in an alternate
host plant. However, the conversion rate of CBGA to THCA in tobacco hairy root
cultures was limited to 8.2% (Taura 2009 ).
16.4.3.2 Over-Expression of Tetrahydrocannabinolic Acid Synthase
in Yeast Cultures
Despite the success of transgenic hairy root and insect cell cultures (see
Sect.16.4.2.1) for functionalTHCASexpression, these systems have limitations
impeding their suitability for the production of THC. The hairy root expression
system demonstrated a low rate of THCA synthesis (Taura 2009 ) and insect cell
cultures require an expensive complex medium and elaborate viral infection and
354 M. Feeney and Z.K. Punja