title: Δ9-Tetrahydrocannabinolic acid synthase: The application of a plant secondary metabolite enzyme in biocatalytic chemical synthesis.
abstract: Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from the secondary metabolism of Cannabis sativa L. catalyzes the oxidative formation of an intramolecular CC bond in cannabigerolic acid (CBGA) to synthesize Δ(9)-tetrahydrocannabinolic acid (THCA), which is the direct precursor of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Aiming on a biotechnological production of cannabinoids, we investigated the potential of the heterologously produced plant oxidase in a cell-free system on preparative scale. THCAS was characterized in an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate and to allow in situ product removal. Compared to the single phase aqueous setup the specific activity decreased by a factor of approximately 2 pointing to a substrate limitation of CBGA in the two-liquid phase system. However, the specific activity remained stable for at least 3h illustrating the benefit of the two-liquid phase setup. In a repeated-batch setup, THCAS showed only a minor loss of specific activity in the third batch pointing to a high intrinsic stability and high solvent tolerance of the enzyme. Maximal space-time-yields of 0.121gL(-1)h(-1) were reached proving the two-liquid phase concept suitable for biotechnological production of cannabinoids.
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: D002188:Cannabis / Q000201:enzymology* / Q000235:genetics; D013759:Dronabinol / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D013759:Dronabinol; D019746:Intramolecular Oxidoreductases; C100677:delta(1)-tetrahydrocannabinolic acid synthase; C025351:delta(9)-tetrahydrocannabinolic acid
keywords: Biocatalysis; Cannabinoids; Natural product synthesis; Oxidase; Two-liquid phase setup