Acknowledgements

THCA Synthase

· Soluble, monomeric flavoenzyme from Cannabis sativa

· Catalyses the oxidative cyclization of CBGA to THCA

· Crystal structure proposed

· Activity dependent on covalently bound co-factor FAD (blue)

· Disulfide bond (Cys37 and Cys99) and 6 N-linked glycosylation sites

· Recombinant expression of enzyme successful in hairy roots, Sf9 insect cells

and P. pastoris [3],[4],[5]

· THCAS functionally expressed in P. pastoris cells (63.5 fold higher than reported so far)

· THCAS targeted into cell vacuoles

· Highest THCAS activity of 145 pkat/mL after 192 h of incubation at 15 °C

· 168 g CDW/L (OD200) produce at least 2.5 mM THCA (0.9 g THCA/L) after 1.75 h of incubation at 52 °C

References

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

Technische Biochemie, Fakultät Bio- und Chemieingenieurwesen, Technische Universität Dortmund

Background and Objectives

[1]: Taura F. et al., 2007, Biochemical and biophysical research communications, 361(3):675-680[2]: Mechoulam R., 1970, Science, 168:1159–1166.[3]: Sirikantaramas S. et al., 2007, Current Pharmaceutical Biotechnology, 8(4):237-243[4]: Shoyama Y. et al., 2005, Acta crystallographica, 61(8):799-801[5]: Shoyama Y. et al., 2012, Journal of molecular biology, 423(1):96-105[6]: Zirpel B. et al., 2015, Biotechnology letters, DOI 10.1007/s10529-015-1853-x

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. Studies were conducted with the permission of

No. 4584989 issued by the Federal Institute for Drugs and Medical Devices (BfArM), Germany.

Summary Future Prospects

· Increasing pharmaceutical demand for

Δ9-tetrahydrocannabinol (THC)

· Legislation prevents growing of cannabis

· Chemical synthesis and extraction from plant proves problematic [1],[2]

· Final goal is production of Δ9-tetrahydrocannabinolic acid (THCA), precursor of THC, by a microbial host at industrial relevant scale

· Aim of this study: THCA synthase, catalyzing final step of THCA synthesis, is functionally expressed in yeasts to investigate the possible application of a whole cell production system for THCA

· Shaking flask cultivations; modified from [1]

· Expression temperatures tested: 10 °C, 15 °C, 20 °C, 25 °C

· THCAS expression highest at 15 °C

· 100 pkat/mL intracellular after 192 h

· 45 pkat/mL extracellular after 192 h

THCAS Expression

Biosynthesis of THCA

Strategy· Low water solubility of substrate CBGA, denaturing properties of H2O2 produced during

regeneration of cofactor FAD and possibility to link THCAS activity to preceding enzymatic step catalysed by the integral membrane protein CBGAS favor a whole cell approach over a cell free system

· Intracellular expression (PEP4 signal peptide in yeasts) was investigated using Pichia pastoris, Saccharomyces cerevisiae and also Escherichia coli (modified strain able to form disulfide bonds in the cytosol)

· P. pastoris proved to be the most suitable expression host while no active THCAS was obtained from E. coli cultures

Whole Cell Catalysis

· The solubility issues in a biotechnological THCA production can be circumvented by employing P. pastoris whole cells

· Coupling with integral membrane protein CBGAS or soluble enzyme catalyzing equivalent reaction possible

· THCA production might be coupled to enzyme expression during cell growth to prolong catalytic activity

Experimental design:

· Whole cell catalysis performed at 37 °C and 52 °C using cells expressing THCAS for 192 h at 15 °C

· Cells were resuspended in 100 mM Na-citrate buffer pH 5.5

· Cell viability with and without CBGA investigated over time of catalysis

· CBGA fed to the cells (indicated by arrows)

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Exp

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Temperature Dependency

native host

yeast host

fermentationproduct

Transfer of biosynthetic pathway

O

OH

COOH

· THCAS activity of cell lysate supernatant determined at different temperatures

· Activity measured in 100 mM Na-citrate buffer pH 5.5

· THCAS temperature optimum at 52 °C

· THCAS activity assay for analysis of expression cultures performed at 37 °C (60 % of maximum activity)

Results:

· Data indicate accumulation of CBGA and THCA in cell membranes (not shown)

· 42 g CDW/L produce 1 mM of THCA, independent from cell viability and temperature (Fig. 6 and 7)

· Data indicate enzyme inactivation due to e.g. H2O2 production after a certain substrate turnover

· Production is up-scalable, but dependent on employed enzyme amount (Fig. 8)

Fig. 4: THCAS expression in P. pastoris shaking flask cultures at 15 °C; taken from [6] Fig. 5: Temperature dependency of THCAS activity; taken from [6]

Fig. 1: General idea for alternative THCA production

Fig. 2: Detail of the biosynthetic pathway for the THCA production in Cannabis sativa; modified from [3]

Fig. 3: 3D-structure model of the THCA synthase; cofactor FAD in blue; modified from [5]

Fig. 6: THCA production by 42 g CDW/L whole cells of P. pastoris at 37 °C and 52 °C; starting concentration of CBGA was 1 mM; arrows indicate addition of 1 mM CBGA; taken from [6]

Fig. 7: Cell viability of P. pastoris cells during whole cell catalysis at 37 °C and 52 °C; Cells were also incubated without CBGA; taken from [6]

Fig. 8: Scalability of THCA production using P. pastoris whole cells; catalysis performed at 52 °C

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