Production of [Delta]9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing [Delta]9-tetrahydrocannabinolic acid synthase from Cannabis sativa l

Objective The [Delta]9-tetrahydrocannabinolic acid synthase (THCAS) from Cannabis sativa was expressed intracellularly in different organisms to investigate the potential of a biotechnological production of [Delta]9-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^sup -1^ (intracellular) and 44 pkat ml^sup -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^sup -1^) THCA could be produced by 10.5 g^sub CDW^ l^sup -1^ before enzyme activity was lost. Conclusion Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production