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.
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.
Whole cells of P. pastoris offer the capability of synthesizing pharmaceutical THCA production
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This study was financially supported by the Graduate Cluster Industrial Biotechnology (CLIB). The authors are thankful to the thesis students for their excellent help during the laboratory work: Dirk Münker, David Dannheisig and Madeleine Dorsch. We are also grateful to Parijat Kusari for critically reading this manuscript. Studies were conducted with the permission of No. 4584989 issued by the Federal Institute for Drugs and Medical Devices (BfArM), Germany.
Supplementary Table 1: List of microorganisms used for expression of THCAS.
Supplementary Table 2: List of plasmids.
Supplementary Fig. 1: Screening of P. pastoris clones—volumetric THCAS activity; cultures were inoculated at 0.105 gCDW l−1. Cultures were grown at 200 rpm and 20 °C. Methanol was added every 24 h at 0.5 % (v/v). Values are calculated from biological duplicates.
Supplementary Fig. 2: Screening of P. pastoris clones—specific THCAS activity; cultures were inoculated at 0.105 gCDW l−1. Cultures were grown at 200 rpm and 20 °C. Methanol was added every 24 h at 0.5 % (v/v). Values are calculated from biological duplicates.
Supplementary Fig. 3: Expression of THCAS using PP2_HC; Cultures were grown in 3-baffled shake-flasks at 200 rpm and 10 °C. Methanol was added every 24 h at 0.5 % (v/v). Data points represent the means of three biological replicates with two technical replicates and error bars represent the standard deviation.
Supplementary Fig. 4: Expression of THCAS using PP2_HC; Cultures were grown in 3-baffled shake-flasks at 200 rpm and 20 °C. Methanol was added every 24 h at 0.5 % (v/v). Data points represent the means of three biological replicates with two technical replicates and error bars represent the standard deviation.
Supplementary Fig. 5: Expression of THCAS using PP2_HC; Cultures were grown in 3-baffled shake-flasks at 200 rpm and 25 °C. Methanol was added every 24 h at 0.5 % (v/v). Data points represent the means of three biological replicates with two technical replicates and error bars represent the standard deviation.
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Zirpel, B., Stehle, F. & Kayser, O. Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa l.. Biotechnol Lett 37, 1869–1875 (2015). https://doi.org/10.1007/s10529-015-1853-x
- Cannabigerolic acid
- Cannabis sativa
- Pichia pastoris
- Δ9-Tetrahydrocannabinolic acid
- Whole cell bioconversion