dinucleotide (FAD) cofactor for its activity and the enzyme releases hydrogen
peroxide as a by-product of the reaction.
16.4.2.2 Identification of Enzymes Involved in the Biosynthesis
of Olivetolic Acid
Thefirst committed step toward the synthesis of THCA is the generation of
olivetolic acid (OLA) from hexanoyl-CoA and three molecules of malonyl-CoA
(Marks et al. 2009 ; Fig.16.1). Based on the structure of OLA, the reaction is
expected to be catalysed by a member of the polyketide synthase (PKS) family,
sometimes referred to as OLS (Raharjo et al. 2004 ; Taura et al. 2009 ; see
Sect.16.4.1.2). Several groups have sought to identify the PKS enzyme responsible
for synthesizing OLA (Raharjo et al. 2004 ; Marks et al. 2009 ; Taura et al. 2009 ).
RecombinantPKSgene candidates were constructed from cDNA isolated from
Cannabis tissues. To obtain a sufficient amount of protein for biochemical char-
acterization, recombinantPKSgenes were transformed intoE. colifor expression
and were purified by affinity chromatography. Biochemical analyses carried out by
all groups led to inconclusive results; OLA was not determined to be a product of
assays involving the candidate PKS enzymes (Raharjo et al. 2004 ; Marks et al.
2009 ; Taura et al. 2009 ). Gagne et al. ( 2012 ) solved the mystery by identifying
olivetolic acid cyclase (OAC) as an accessory enzyme that functions cooperatively
with PKS to form OLA. Recombinant CsOACgene candidates were
over-expressed inE. coliand purified. Biochemical assays identified one OAC
candidate that formed OLA when assayed in combination with PKS. To determine
their subcellular location, genes encoding both enzymes were fused to genes
encodingfluorescent proteins and the fusions were transiently expressed in
N. benthamianaby agroinfiltration. Confocal microscopy revealed that PKS-CFP
and OAC-YFP were co-localized to the same cellular compartment, the cytoplasm,
suggesting that they are physically capable of interacting or sharing reaction
products. To demonstrate OAC activity in vivo, Gagne et al. ( 2012 ) generated
transgenic yeast cells over-expressingCsPKSandCsOAC. When fed with a hex-
anoate precursor, yeast secreted OLA into the culture medium (Fig.16.1).
16.4.3 Metabolic Engineering of the Cannabinoid Pathway
in Heterologous Hosts
The demand for cannabinoids is increasing but there are limitations to how much
can be supplied byC. sativa(Zirpel et al. 2015 ). THC is commercially available but
extremely expensive (Taura et al. 2007 ). Cannabis is capable of synthesizing large
amounts of THCA and CBDA (the precursors to THC and CBD, respectively) but
extracts are composed of a complex mixture of compounds that are difficult to
16 The Role ofAgrobacterium-Mediated and Other Gene-Transfer... 353