title: Δ(9)-Tetrahydrocannabinolic acid synthase production in Pichia pastoris enables chemical synthesis of cannabinoids.
abstract: Δ(9)-Tetrahydrocannabinol (THC) is of increasing interest as a pharmaceutical and bioactive compound. Chemical synthesis of THC uses a laborious procedure and does not satisfy the market demand. The implementation of biocatalysts for specific synthesis steps might be beneficial for making natural product availability independent from the plant. Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from C. sativa L. catalyzes the cyclization of cannabigerolic acid (CBGA) to Δ(9)-tetrahydrocannabinolic acid (THCA), which is non-enzymatically decarboxylated to THC. We report the preparation of THCAS in amounts sufficient for the biocatalytic production of THC(A). Active THCAS was most efficiently obtained from Pichia pastoris. THCAS was produced on a 2L bioreactor scale and the enzyme was isolated by single-step chromatography with a specific activity of 73Ug(-1)total protein. An organic/aqueous two-liquid phase setup for continuous substrate delivery facilitated in situ product removal. In addition, THCAS activity in aqueous environments lasted for only 20min whereas the presence of hexane stabilized the activity over 3h. In conclusion, production of THCAS in P. pastoris Mut(S) KM71 KE1, subsequent isolation, and its application in a two-liquid phase setup enables the synthesis of THCA on a mg scale.
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical & Chemical Engineering, TU Dortmund University, Dortmund, Germany; Department of Solar Materials, Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Lange
authors.affiliation: Department of Solar Materials, Helmholtz Centre for Environmental Research (UFZ), Leipzig, Germany. Electronic address: andreas.schmid@ufz.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Schmid
authors.affiliation: Laboratory of Chemical Biotechnology, Department of Biochemical & Chemical Engineering, TU Dortmund University, Dortmund, Germany.
authors.forename: Mattijs K
authors.initials: MK
authors.lastname: Julsing
mesh_terms: D061249:Batch Cell Culture Techniques; D001565:Benzoates / Q000378:metabolism; D055162:Biocatalysis; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D002186:Cannabinoids / Q000138:chemical synthesis*; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D019746:Intramolecular Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001565:Benzoates; D002186:Cannabinoids; C100679:cannabigerolic acid; D013759:Dronabinol; D019746:Intramolecular Oxidoreductases; C025351:delta(9)-tetrahydrocannabinolic acid
keywords: Biocatalysis; Cannabinoids; Oxidase; Oxidative C–C bond formation; Two-liquid phase setup