Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis satival.
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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
KeywordsCannabigerolic acid Cannabis sativa Pichia pastoris Δ9-Tetrahydrocannabinolic acid Synthase Whole cell bioconversion
Since a scale-up approach with the isolated THCAS is not suitable due to: (i) the low water solubility of the substrate CBGA, (ii) the denaturing properties of H2O2, and (iii) the integral membrane enzyme CBGA synthase, that cannot be secreted and therefore not feasibly implemented into a cell-free production system, we focused in this study on the intracellular THCAS expression in Escherichia coli, Saccharomyces cerevisiae and P. pastoris cells and the possible application towards a whole cell production system of THCA.
Materials and methods
Δ9-THCA was purchased from THC Pharm (Frankfurt am Main, Germany). Cannabigerolic acid (CBGA) was purchased from Taros Chemicals (Dortmund, Germany).
Microorganisms, genes and plasmids
Cloning strategies and a detailed list of all strains and plasmids used in this study are given in the supplementary material (Supplementary Tables 1, 2). Synthetic coding sequences of THCAS (GenBank accession number AB057805) were codon optimized for expression in Saccharomyces cerevisiae and Pichia pastoris and purchased without signal peptide from GeneArt (Regensburg, Germany). Recombinant expression of THCAS was conducted with the following microorganisms: E. coli SHuffle T7 Express and SHuffle T7 Express lysY (NEB, Frankfurt am Main, Germany), containing modifications to enable disulfide bond formation in the cytosol, were used with pET28a(+) and pET32a(+) vectors (Merck, Darmstadt, Germany); Saccharomyces cerevisiae CEN.PK2-1CΔgal1 (Euroscarf, Frankfurt am Main, Germany) deficient in the β-galactokinase and CEN.PK2-1CΔgal1Δpep4 (this study) additionally deficient in the vacuolar Proteinase A; Pichia pastoris PichiaPink strains 1, 2 and 3 (Invitrogen, Darmstadt, Germany). All Pichia strains are adenine auxotrophs. Strain 2 and 3 contain additional knockouts of the vacuolar proteases Proteinase A (pep4) and Proteinase B (prb1), respectively. Pichia strains were transformed with linearized high copy (pPink_HC_THCAS) and low copy (pPink_LC_THCAS) vectors for integration into chromosomal TRP2 gene and Saccharomyces strains were transformed with pDionysos_THCAS vector, all containing a cDNA of THCAS with three additional histidines at the C-terminus and an additional N-terminal sequence coding for a 24 aa signal peptide from Proteinase A [UniProt accession number F2QUG8 in pPink vectors (Invitrogen, Darmstadt, Germany) and P07267 in pDionysos vector (Stehle et al. 2008)] for targeting into the cell vacuoles.
Detailed media compositions are described in Supporting Information. If not stated otherwise, cells were cultivated as follows. Recombinant E.coli cells were grown in 1 l flasks, containing 100 ml LB-medium (50 µg kanamycin ml−1, 33 µg chloramphenicol ml−1, 100 µg spectinomycin ml−1) at 37 °C and 200 rpm to an OD600 of 0.6. THCAS expression was induced by addition of 1 mM IPTG and cells grown for 16 h at 20 °C. Recombinant S.cerevisiae cells were grown in minimal medium without leucine at 30 °C and 200 rpm for 24 h. Cells were used to inoculate 100 ml of 2 × YPAD medium at an OD600 of 0.5 and incubated after induction with 0.5 % (w/v) galactose at 20 °C and 200 rpm for 144 h. Recombinant P. pastoris cells were grown in BMGY at 30 °C and 200 rpm for 24 h. Afterwards, cells were harvested by centrifugation at 5000×g for 5 min and resuspended in modified BMMY (mBMMY) (Taura et al. 2007) to an OD600 of 20. Finally, Pichia cells were cultivated at 15 °C and 200 rpm until no increase in THCAS activity could be observed and supplemented with 0.5 % (v/v) methanol every 24 h for induction of protein expression.
Cell density was measured from the OD600 value and cell dry mass (CDW) was calculated according to Tolner et al. (2006) with a correlation of CDW (g l−1) = 0.21 g l−1 × OD600. Protein concentrations were measured using Bradford assay (Ernst and Zor 2010). Methanol concentrations were determined by HPLC–UV analysis at 210 nm using Hi-Plex H 300 × 7.7 mm column by isocratic elution (5 mM H2SO4 in H2O) at 0.5 ml min−1 at 65 °C.
THCAS activity assay
A detailed protocol for cell lysis is described in the Supporting Information. Briefly, cells were harvested by centrifugation. Cell pellets were resuspended in 100 mM sodium citrate buffer pH 5.5 and the supernatant was diluted to 50 % (v/v) with 100 mM sodium citrate buffer pH 5.5. Yeast cells were lysed by glass beads and E.coli cells by sonication. After centrifugation of cell debris, lysate supernatants and diluted culture supernatants were used for determination of THCAS activity at 37 °C by addition of CBGA (final concentration 100 µM, 1 % (v/v) DMSO). Activity assays were stopped by addition of 0.3 assay volumes trifluoroacetic acid and 2.7 assay volumes acetonitrile (ACN) followed by incubation on ice for 15 min. Supernatants were analyzed after centrifugation (13,100×g, 4 °C, 30 min) by HPLC using a Nucleosil 100-5 C18 column. Isocratic elution [25 % (v/v) H2O with 0.1 % (v/v) TFA/75 % (v/v) ACN] was used at 0.7 ml min−1. Identification of CBGA and THCA was performed by HPLC–MS and quantification by HPLC–UV at 225 nm and 35 °C.
Results and discussion
Comparison of THCAS activities of recombinant Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris strains
Comparison of different organisms regarding highest obtained Δ9-tetrahydrocannabinolic acid synthase (THCAS) activity; cultures were inoculated at 0.105 gCDW ml−1 (OD600 of 0.5) and cultivated at 20 °C. Enzymatic activity of lysate supernatant was measured at 37 °C. Values are calculated from biological duplicates
Volumetric activity intracellular (pkat ml−1)
Specific activity intracellular (pkat gCDW−1)
E. coli (all tested strains)
S. cerevisiae CEN.PK2-1C ∆gal1∆pep4 pDionysos_THCAS
1 ± 0.1
5.6 ± 0.3
PichiaPink2 ∆pep4 pPink_HC_THCAS (PP2_HC)
1.4 ± 0.1
12.4 ± 0.9
Optimization of THCAS expression in P. pastoris
Comparison of intracellular and extracellular Δ9-tetrahydrocannabinolic acid synthase (THCAS) activity at different cultivation temperatures; activities of culture or lysate supernatant were measured at 37 °C. The maximum values obtained during each cultivation are shown. Values are calculated from biological triplicates with two technical replicates
Volumetric activity intracellular (pkat ml−1)
Volumetric activity extracellular (pkat ml−1)
Specific activity intracellular (pkat gCDW−1)
Specific activity extracellular (pkat gCDW−1)
54 ± 4
43 ± 3
189 ± 16
152 ± 10
98 ± 5
44 ± 4
405 ± 8
179 ± 17
41 ± 3
25 ± 3
185 ± 9
114 ± 10
14 ± 0.4
7.6 ± 0.7
74 ± 2
41 ± 4
Whole cell bioconversion of cannabigerolic acid (CBGA) to Δ9-tetrahydrocannabinolic acid (THCA) using P. pastoris cells
Preliminary experiments showed a low solubility of CBGA in aqueous solutions, e.g. around 200 µM in 100 mM sodium citrate buffer pH 5.5 (data not shown), impairing the production of higher amounts of THCA using cell-free aqueous systems or purified proteins. On the contrary, an immediate uptake of at least 14 mM CBGA into the cells could be observed (data not shown). Therefore, whole cell bioconversion of CBGA to THCA was investigated. A temperature dependency of THCAS activity in cell lysate supernatant is shown in Fig. 3.
The amount of enzyme used in the bioconversion was able to convert 1 mM CBGA to THCA (0.36 g THCA l−1) before loss of activity arose. As this effect is not temperature-dependent (Fig. 4b), the inactivation of enzyme might be due to H2O2 production upon FAD regeneration. Nevertheless, the concentration of THCA was increased by 400 % compared to reactions with lysate and by 900 % compared to reports from Taura et al. (2007). Increasing the employed amount of cells could yield higher THCA levels. Furthermore, co-expression of a catalase or coupling THCA production to enzyme expression during cell growth could prolong enzymatic activity and thus increase THCA levels.
The expression of THCA synthase from Cannabissativa l. was investigated using prokaryotic and eukaryotic expression systems. While no functional expression could be achieved in E. coli, the highest enzyme activity was obtained in P. pastoris cultures. Under optimized conditions, volumetric THCAS activity levels were increased by 6350 % compared to previous reports (Taura et al. 2007). The solubility issues in a biotechnological THCA production could be circumvented by employing P. pastoris whole cells. Finally, the whole cell bioconversion leads to the production of 1 mM THCA (0.36 g THCA l−1). Thus, in future whole cells might provide an alternative method for the production of pharmaceutical THC.
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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