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.
Taura
Futoshi
F
Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan. taura@phar.kyushu-u.ac.jp
Dono
Emi
E
Sirikantaramas
Supaart
S
Yoshimura
Kohji
K
Shoyama
Yukihiro
Y
Morimoto
Satoshi
S
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