Bastian Zirpel1, Felix Stehle, Oliver Kayser. 1. Laboratory of Technical Biochemistry, Department of Biochemical & Chemical Engineering, TU Dortmund University, Emil-Figge-Str. 66, 44227, Dortmund, Germany.
Abstract
OBJECTIVE: The Δ9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of Δ9-tetrahydrocannabinolic acid (THCA) using whole cells. RESULTS: Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml(-1) (intracellular) and 44 pkat ml(-1) (extracellular) after 192 h of cultivation at 15 °C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l(-1)) THCA could be produced by 10.5 gCDW l(-1) before enzyme activity was lost. CONCLUSION: Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production.
OBJECTIVE: The Δ9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of Δ9-tetrahydrocannabinolic acid (THCA) using whole cells. RESULTS: Functional expression of THCAS was obtained in Saccharomyces cerevisiae and Pichia (Komagataella) pastoris using a signal peptide from the vacuolar protease, proteinase A. No functional expression was achieved in Escherichia coli. The highest volumetric activities obtained were 98 pkat ml(-1) (intracellular) and 44 pkat ml(-1) (extracellular) after 192 h of cultivation at 15 °C using P. pastoris cells. Low solubility of CBGA prevents the THCAS application in aqueous cell-free systems, thus whole cells were used for a bioconversion of cannabigerolic acid (CBGA) to THCA. Finally, 1 mM (0.36 g THCA l(-1)) THCA could be produced by 10.5 gCDW l(-1) before enzyme activity was lost. CONCLUSION: Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production.
Authors: Dennis J Sholler; Marilyn A Huestis; Benjamin Amendolara; Ryan Vandrey; Ziva D Cooper Journal: Pharmacol Biochem Behav Date: 2020-10-18 Impact factor: 3.533
Authors: Leonardo Martins-Santana; Luisa C Nora; Ananda Sanches-Medeiros; Gabriel L Lovate; Murilo H A Cassiano; Rafael Silva-Rocha Journal: Front Bioeng Biotechnol Date: 2018-10-03