amplification procedures, so a more practical expression system was desired (Taura
et al. 2007 ). Attention was then turned to yeast as an alternative expression system.
Thefirst effort to produce THCA in yeast showed promising results using a
Pichia pastoris expression system (Taura et al. 2007 ; Ahmad et al. 2014 ).
P. pastoriscells were transformed with recombinantCsTHCASusing a commercial
kit (Taura et al. 2007 ). Transgenic P. pastoris cells over-expressingTHCAS
secreted most of the enzyme into the culture medium. Supplementing the medium
with the precursor CBGA resulted in only a 10% bioconversion to THCA.
Suspecting that the low conversion rate was a result of the activity of other
cannabinoid-metabolizing enzymes produced by P. pastoris, the cells were
removed from the medium. By feeding CBGA to the culture supernatent, the
conversion rate was increased to 98%. However, low solubility of the CBGA
precursor in the culture medium posed a limitation to the amount of THCA
recovered. Despite this, biosynthesis of THCA using this cell-free system was much
greater than hairy roots. Furthermore, recombinant THCAS purified from
P. pastorishad a much higher level of activity than both the native THCAS purified
from C. sativa and the recombinant THCAS produced by insect cells
(Sirikantaramas et al. 2004 ; Taura et al. 2007 ). Overall, secretion of recombinant
THCAS fromP. pastorisfor use in a cell-free system to convert CBGA to THCA
was encouraging, but had limitations.
Building upon thesefindings, Zirpel et al. ( 2015 ) studied the intracellular
expression of THCAS inP. pastoris, Saccharomyces cerevisiae and E. coli.
TransgenicE. coliharbouring the recombinantCsTHCASgene failed to express the
enzyme and had no detectable THCAS activity. The authors speculate that func-
tional THCAS expression may require eukaryotic chaperones or protein glycosy-
lation, soE. coliwas not a suitable host in this case. On the other hand, the two
transgenic yeast expression systems both expressed THCAS, with the highest
enzyme activity achieved byP. pastoris. To avoid secretion of the enzyme from
yeast cells, THCAS was targeted to the yeast vacuole using a vacuolar signal
peptide. Supplementing CBGA to theP. pastorisculture medium resulted in the
immediate uptake of the precursor by the cells and a high rate of bioconversion to
THCA, which remained embedded in the yeast cell membrane. Using this
whole-cell bioconversion system, Zirpel et al. ( 2015 ) achieved an exponential
increase in THCAS activity levels compared to the cell-free system described by
Taura et al. ( 2007 ). An author of this study, Dr. Oliver Kayser, is extending this
research in collaboration with THC Pharm GmbH (Frankfurt, Germany) to engineer
the complete pathway to THCA in yeast and to scale up the system for industrial
production (Hodgkins 2015 ; Khamsi 2015 ).
16.4.3.3 Metabolic Engineering of the Cannabinoid Pathway
for Commercialization
The prospect of commercializing cannabinoids as therapeutics appears bright
(Brockstein 2016 ). There is great interest in developing Cannabis-based drugs for
16 The Role ofAgrobacterium-Mediated and Other Gene-Transfer... 355