cytosol. These results, along with others, provide support for the role of CsAAE1 as
the hexanoyl-CoA synthetase that supplies hexanoyl-CoA to the cannabinoid
pathway. Analysis ofCsAAE1function using transgenic approaches such as RNAi
silencing was not possible due to the inability to genetically transform Cannabis
(Stout et al. 2012 ).
16.4.2 Enzyme Kinetics: Expression and Purification
of Recombinant Cannabis Enzymes
in Heterologous HostsElucidation of the cannabinoid biosynthetic pathway requires a comprehensive
knowledge of the whole biosynthetic pathway, including a detailed understanding
of the enzymes involved and their function (Oksman-Caldentey and Inze 2004 ;
Zhang et al. 2004 ; Ajikumar et al. 2010 ; Lim et al. 2011 ). This is particularly
important for downstream applications such as the metabolic engineering of
cannabinoid pathways in heterologous hosts (Sirikantaramas et al. 2005 ; Stout et al.
2012 ). Gene transfer technologies have played a valuable role in characterizing
enzyme function by enabling high-level expression of native Cannabis biosynthetic
enzymes in heterologous hosts.
16.4.2.1 Characterization of the Tetrahydrocannabinolic Acid
Synthase Reaction Mechanism
In marijuana cultivars, the cannabinoid pathway enzyme THCAS is highly
expressed (van Bakel et al. 2011 ; Weiblen et al. 2015 ). THCAS catalyses the
formation of the main psychoactive cannabinoid, THCA, from cannabigerolic acid
(CBGA) (Taura et al. 1995 ; Fig.16.1). Therefore, it is an important enzyme con-
trolling the psychoactivity of the plant (Baker et al. 2003 ; Pertwee 2004 ). To further
characterize the mechanism by which THCAS carries out this reaction, biochemical
analyses were performed on purified enzyme. Efforts using the native enzyme were
complicated by the inability to purify enough THCAS from Cannabis extracts and
its subsequent characterization did not provide enough detailed functional and
structural information (Sirikantaramas et al. 2004 ; Taura et al. 2007 ). Therefore,
THCAS was over-expressed in insect cells that were transformed using a bac-
ulovirus carrying the recombinantCsTHCASgene (Sirikantaramas et al. 2004 ).
Baculoviruses are versatile vectors that transfer recombinant genes into insect and
mammalian cells for the production of recombinant proteins (Kost et al. 2005 ). The
baculovirus-insect expression system achieved high levels of recombinant THCAS
production that possessed activity after purification (Sirikantaramas et al. 2004 ).
Biochemical analyses on the purified recombinant protein demonstrated that
THCAS is an oxidase that is dependent on a covalently attachedflavin adenine
352 M. Feeney and Z.K. Punja