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Archives of Industrial Biotechnology | Volume 2
May 14-15, 2018 | Montreal, Canada
World Yeast Congress
T
hecannabinoidsynthaseenzymesTetrahydrocannabinolic
acid (THCA) synthase and Cannabidiolic acid (CBDA)
synthase were recombinantly produced in yeast
Pichia
pastoris
Mut+ strain. The coding regions of THCA synthase
and CBDA synthase genes were codon optimized for
Pichia
expression. Both synthase genes were operably linked to
the methanol inducible AOX1 promoter, an N-terminal alpha
mating factor secretion signal, and a C-terminal 6x His-tag.
These elements provide for inducible expression of the genes
and simple processing/purification of the encoded enzymes.
Each synthase constructwas cloned into Invitrogen’s pPIC3.5K
plasmid. The recombinant plasmids were transformed into
Pichia
strain GS-115.
Pichia
clones transformed with multiple
copies of each construct were selected based on their
resistance to varying amount of geneticin concentrations.
Gene copy numbers were further verified with RT-PCR.
Fermentation conditions were optimized by investigating the
impact of pH, temperature, methanol feed, and fermentation
medium composition on cell growth and enzyme yield. The
fermentation conditions were further optimized in a pilot
scale, 14-liter fermenter. Based on these results, production
was successfully scaled up to 500-liter fermenters. The
Teewinot enzyme production system produced active THCA
synthase and CBDA synthase enzymes. The THCA synthase
converts chemically synthesized CBGA into
∆
9-THCA and
CBCA or chemically synthesized Cannabigerovarinic acid
(CBGVA) into Tetrahydrocannabivarin (THCVA) and CBCVA in
a bioreactor. The ratio of
∆
9-THCA to CBCA and THCVA to
CBCVA is dependent on reaction conditions including pH. The
CBDA synthase enzyme converted chemically synthesized
CBGA into CBDA, CBCA, and THCA or chemically synthesized
CBGVA into CBDVA, CBCVA, and THCVA. Once again, the
molar ratios of CBDA, CBCA, and THCA or the molar ratios
of CBDVA, CBCVA, and THCVA produced in the bioreactor
were dependent on reaction conditions such as pH. Each
of the biocatalytically-produced cannabinoids was purified
to greater than 99.5% purity. The identity and structure of
each biocatalytically-produced cannabinoid was confirmed
by HPLC, mass spectral, and NMR analysis.
Speaker Biography
Mingyang Sun has completed his Master’s Degree in Synthetic Biology from Concordia
University, Montreal. He is a Co-inventor of several US patents on cannabinoid
biosynthesis and the Vice President of Teewinot Laboratories Inc, subsidiary of
Teewinot Life Sciences Corporation.
e:
ming@tlscorp.comCannabinoids biosynthesis using recombinant cannabinoid synthase enzymes expressed from
industrial
yeast Pichia pastoris
Mingyang Sun, Oleg Tyurin, Jasleen Bains, Neil Von Wittgenstein, Richard Peet
and
Malcolm Kavarana
Teewinot Life Sciences Corporation, USA