Production of Delta(1)-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris.
Delta(1)-Tetrahydrocannabinolic acid (THCA) synthase is the enzyme that catalyzes the oxidative cyclization of cannabigerolic acid into THCA, the acidic precursor of Delta(1)-tetrahydrocannabinol. We developed a novel expression system for THCA synthase using a methylotrophic yeast Pichia pastoris as a host. Under optimized conditions, the transgenic P. pastoris secreted approximately 1.32nkat/l of THCA synthase activity, and the culture medium, from which the cells were removed, effectively synthesized THCA from cannabigerolic acid with a approximately 98% conversion rate. The secreted THCA synthase was readily purified to homogeneity. Interestingly, endoglycosidase treatment afforded a deglycosylated THCA synthase with more catalytic activity than that of the glycosylated form. The non-glycosylated THCA synthase should be suitable for structure-function studies because it displayed much more activity than the previously reported native enzyme from Cannabis sativa as well as the recombinant enzyme from insect cell cultures.
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan. taura@phar.kyushu-u.ac.jp
Futoshi
F
Taura
Emi
E
Dono
Supaart
S
Sirikantaramas
Kohji
K
Yoshimura
Yukihiro
Y
Shoyama
Satoshi
S
Morimoto
D001565:Benzoates / Q000378:metabolism; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors; D019076:Transgenes
D001565:Benzoates; D011994:Recombinant Proteins; C524353:delta(1)-tetrahydrocannabinolic acid; C100679:cannabigerolic acid; D013759:Dronabinol; D019746:Intramolecular Oxidoreductases; C100677:delta(1)-tetrahydrocannabinolic acid synthase