Advertisement

Biotechnology Letters

, Volume 37, Issue 9, pp 1869–1875 | Cite as

Production of Δ9-tetrahydrocannabinolic acid from cannabigerolic acid by whole cells of Pichia (Komagataella) pastoris expressing Δ9-tetrahydrocannabinolic acid synthase from Cannabis sativa l.

  • Bastian Zirpel
  • Felix Stehle
  • Oliver Kayser
Original Research Paper

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

Keywords

Cannabigerolic acid Cannabis sativa Pichia pastoris Δ9-Tetrahydrocannabinolic acid Synthase Whole cell bioconversion 

Notes

Acknowledgments

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.

Supporting information

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.

Supplementary material

10529_2015_1853_MOESM1_ESM.docx (928 kb)
Supplementary material 1 (DOCX 928 kb)

References

  1. Carlini EA (2004) The good and the bad effects of (-) trans-delta-9-tetrahydrocannabinol (Delta 9-THC) on humans. Toxicon 44:461–467CrossRefPubMedGoogle Scholar
  2. Delic M, Graf A, Koellensperger G et al (2014) Overexpression of the transcription factor Yap1 modifies intracellular redox conditions and enhances recombinant protein secretion. Microb Cell 1:376–386CrossRefGoogle Scholar
  3. Ernst O, Zor T (2010) Linearization of the bradford protein assay. J Vis Exp 38:1–6Google Scholar
  4. Mechoulam R (1970) Marihuana Chemistry. Science 168:1159–1165CrossRefPubMedGoogle Scholar
  5. Pertwee RG (2006) Cannabinoid pharmacology: the first 66 years. Br J Pharmacol 147:S163–S171Google Scholar
  6. Rothman J, Stevens TH (1986) Protein sorting in yeast: mutants defective in vacuole biogenesis mislocalize vacuolar proteins into the late secretory pathway 47:1041–1051Google Scholar
  7. Shoyama Y, Tamada T, Kurihara K et al (2012) Structure and function of ∆1-tetrahydrocannabinolic acid (THCA) synthase, the enzyme controlling the psychoactivity of Cannabis sativa. J Mol Biol 423:96–105CrossRefPubMedGoogle Scholar
  8. Sirikantaramas S, Morimoto S, Shoyama Y et al (2004) The gene controlling marijuana psychoactivity: molecular cloning and heterologous expression of Delta1-tetrahydrocannabinolic acid synthase from Cannabis sativa L. J Biol Chem 279:39767–39774CrossRefPubMedGoogle Scholar
  9. Stehle F, Stubbs MT, Strack D, Milkowski C (2008) Heterologous expression of a serine carboxypeptidase-like acyltransferase and characterization of the kinetic mechanism. FEBS J 275:775–787CrossRefPubMedGoogle Scholar
  10. Taura F, Dono E, Sirikantaramas S et al (2007) Production of Delta(1)-tetrahydrocannabinolic acid by the biosynthetic enzyme secreted from transgenic Pichia pastoris. Biochem Biophys Res Commun 361:675–680CrossRefPubMedGoogle Scholar
  11. Tolner B, Smith L, Begent RHJ, Chester KA (2006) Production of recombinant protein in Pichia pastoris by fermentation. Nat Protoc 1:1006–1021CrossRefPubMedGoogle Scholar
  12. Trost BM, Dogra K (2007) Synthesis of (-)-Delta9-trans-tetrahydrocannabinol: stereocontrol via Mo-catalyzed asymmetric allylic alkylation reaction. Org Lett 9:861–863PubMedCentralCrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  1. 1.Laboratory of Technical Biochemistry, Department of Biochemical & Chemical EngineeringTU Dortmund UniversityDortmundGermany

Personalised recommendations