Moving cannabinoid production away from the vagaries of plant extraction and into engineered microbes could provide a consistent, purer, cheaper and environmentally benign source of these important therapeutic molecules, but microbial production faces notable challenges. An alternative to microbes and plants is to remove the complexity of cellular systems by employing enzymatic biosynthesis. Here we design and implement a new cell-free system for cannabinoid production with the following features: (1) only low-cost inputs are needed; (2) only 12 enzymes are employed; (3) the system does not require oxygen and (4) we use a nonnatural enzyme system to reduce ATP requirements that is generally applicable to malonyl-CoA-dependent pathways such as polyketide biosynthesis. The system produces ~0.5 g l−1 cannabigerolic acid (CBGA) or cannabigerovarinic acid (CBGVA) from low-cost inputs, nearly two orders of magnitude higher than yeast-based production. Cell-free systems such as this may provide a new route to reliable cannabinoid production.
Your institute does not have access to this article
Subscribe to Nature+
Get immediate online access to the entire Nature family of 50+ journals
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Data will be made available upon request. Materials will be made available with applicable material transfer agreement.
Turner, S. E., Williams, C. M., Iversen, L. & Whalley, B. J. Molecular pharmacology of phytocannabinoids. Prog. Chem. Org. Nat. Prod. 103, 61–101 (2017).
Schrot, R. J. & Hubbard, J. R. Cannabinoids: medical implications. Ann. Med. 48, 128–141 (2016).
Vučković, S., Srebro, D., Vujović, K. S., Vučetić, Č. & Prostran, M. Cannabinoids and pain: new insights from old molecules. Front Pharm. 9, 1259–1259 (2018).
Scherma, M. et al. New perspectives on the use of cannabis in the treatment of psychiatric disorders. Medicines 5, 107 (2018).
Wargent, E. T. et al. The cannabinoid Δ(9)-tetrahydrocannabivarin (THCV) ameliorates insulin sensitivity in two mouse models of obesity. Nutr. Diabetes 3, e68–e68 (2013).
Zamberletti, E. et al. Cannabidivarin treatment ameliorates autism-like behaviors and restores hippocampal endocannabinoid system and glia alterations induced by prenatal valproic acid exposure in rats. Front. Cell. Neurosci. 13, 367 (2019).
Carvalho, Â., Hansen, E. H., Kayser, O., Carlsen, S. & Stehle, F. Designing microorganisms for heterologous biosynthesis of cannabinoids. FEMS Yeast Res. 17, fox037 (2017).
Luo, X. et al. Complete biosynthesis of cannabinoids and their unnatural analogues in yeast. Nature 567, 123–126 (2019).
ElSohly, M. A., Radwan, M. M., Gul, W., Chandra, S. & Galal, A. Phytochemistry of Cannabis sativa L. in Phytocannabinoids: Unraveling the Complex Chemistry and Pharmacology of Cannabis sativa (eds. Kinghorn, A. D., Falk, H., Gibbons, S. & Kobayashi, J.) 1–36 (Springer International Publishing, 2017); https://doi.org/10.1007/978-3-319-45541-9_1
Dolgin, E. The bioengineering of cannabis. Nature 572, S5–S7 (2019).
Degenhardt, F., Stehle, F. & Kayser, O. in Handbook of Cannabis and Related Pathologies (ed. Preedy, V. R.) Ch. 2, 13–23 (Academic Press, 2017); https://doi.org/10.1016/B978-0-12-800756-3.00002-8
Keasling, J. D. Synthetic biology and the development of tools for metabolic engineering. Metab. Eng. 14, 189–195 (2012).
Hodgman, C. E. & Jewett, M. C. Cell-free synthetic biology: thinking outside the cell. Metab. Eng. 14, 261–269 (2012).
Kwok, R. Five hard truths for synthetic biology. Nature 463, 288–290 (2010).
Ricca, E., Brucher, B. & Schrittwieser, J. H. Multi-enzymatic cascade reactions: overview and perspectives. Adv. Synth. Catal. 353, 2239–2262 (2011).
Huffman, M. A. et al. Design of an in vitro biocatalytic cascade for the manufacture of islatravir. Science 366, 1255–1259 (2019).
Valliere, M. A. et al. A cell-free platform for the prenylation of natural products and application to cannabinoid production. Nat. Commun. 10, 565 (2019).
Garcia-Ochoa, F. & Gomez, E. Bioreactor scale-up and oxygen transfer rate in microbial processes: an overview. Biotechnol. Adv. 27, 153–176 (2009).
Lund, S., Hall, R. & Williams, G. J. An Artificial pathway for isoprenoid biosynthesis decoupled from native hemiterpene metabolism. ACS Synth. Biol. 8, 232–238 (2019).
Chatzivasileiou, A. O., Ward, V., Edgar, S. M. & Stephanopoulos, G. Two-step pathway for isoprenoid synthesis. Proc. Natl Acad. Sci. USA 116, 506–511 (2019).
Ward, V. C. A., Chatzivasileiou, A. O. & Stephanopoulos, G. Cell free biosynthesis of isoprenoids from isopentenol. Biotechnol. Bioeng. 116, 3269–3281 (2019).
Crans, D. C. & Whitesides, G. M. A convenient synthesis of disodium acetyl phosphate for use in in situ ATP cofactor regeneration. J. Org. Chem. 48, 3130–3132 (1983).
Gagne, S. J. et al. Identification of olivetolic acid cyclase from Cannabis sativa reveals a unique catalytic route to plant polyketides. Proc. Natl Acad. Sci. USA 109, 12811–12816 (2012).
Taura, F. et al. Characterization of olivetol synthase, a polyketide synthase putatively involved in cannabinoid biosynthetic pathway. FEBS Lett. 583, 2061–2066 (2009).
Tan, Z., Clomburg, J. M. & Gonzalez, R. Synthetic pathway for the production of olivetolic acid in Escherichia coli. ACS Synth. Biol. 7, 1886–1896 (2018).
Flamholz, A., Noor, E., Bar-Even, A. & Milo, R. eQuilibrator—the biochemical thermodynamics calculator. Nucleic Acids Res. 40, D770–D775 (2012).
Chohnan, S., Akagi, K. & Takamura, Y. Functions of malonate decarboxylase subunits from Pseudomonas putida. Biosci. Biotechnol., Biochem. 67, 214–217 (2003).
Koo, J. H. & Kim, Y. S. Functional evaluation of the genes involved in malonate decarboxylation by Acinetobacter calcoaceticus. Eur. J. Biochem. 266, 683–690 (1999).
Stout, J. M., Boubakir, Z., Ambrose, S. J., Purves, R. W. & Page, J. E. The hexanoyl-CoA precursor for cannabinoid biosynthesis is formed by an acyl-activating enzyme in Cannabis sativa trichomes. Plant J. 71, 353–365 (2012).
Peters, Jnr, T. All About Albumin: Biochemistry, Genetics, and Medical Applications (Academic Press, 1995).
Zielinski, K., Sekula, B., Bujacz, A. & Szymczak, I. Structural investigations of stereoselective profen binding by equine and leporine serum albumins. Chirality 32, 334–344 (2020).
Bowie, J. U. et al. Synthetic biochemistry: the bio-inspired cell-free approach to commodity chemical production. Trends Biotechnol. 38, 766–778 (2020).
Kearsey, L. J. et al. Structure of the Cannabis sativa olivetol-producing enzyme reveals cyclization plasticity in type III polyketide synthases. FEBS J. 287, 1511–1524 (2020).
Korman, T. P., Opgenorth, P. H. & Bowie, J. U. A synthetic biochemistry platform for cell free production of monoterpenes from glucose. Nat. Commun. 8, 15526 (2017).
Goldenzweig, A. et al. Automated structure- and sequence-based design of proteins for high bacterial expression and stability. Mol. Cell 70, 380 (2018).
Shoyama, Y., Hirano, H., Makino, H., Umekita, N. & Nishioka, I. Cannabis X: the isolation and structures of four new propyl cannabinoid acids, tetrahydrocannabivarinic acid, cannabidivarinic acid, cannabichromevarinic acid and cannabigerovarinic acid, from Thai cannabis, ‘Meao variant’. Chem. Pharm. Bull. 25, 2306–2311 (1977).
This work was supported by Department of Energy grant nos. DE-FC02-02ER63421 and DE-AR0000556 to J.U.B.
J.U.B. and T.P.K. have founded a company Invizyne Technologies, Inc. to develop cell-free production methods. M.A.V. is also a stockholder.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The concentration of CsOLS vs Product Specificity is plotted at three different AAE3 concentrations. As the concentration of CsOLS or CsAAE3 increase, we observe a decrease in product specificity. Total Peak Area is calculated as the sum of all peaks that appear in the HPLC trace due to the activity of OLS and AAE3. The data points reflect the mean and the error bars represent standard deviation of biological replicates.
The remaining activity in the presence of 5 mM OA (teal bars) or 0.25 mM OA (blue bars). The bar heights reflect the mean and the error bars represent standard deviation of biological replicates. The results of each experiment are shown by the open circles.
The RpMatB reaction system was used to generate OA, which can then be prenylated by the added GPP, catalyzed by NphBM31S. We observe that increasing GPP leads to a decrease in overall production of OA and CBGA, indicating that GPP inhibits the OA pathway. The data points reflect the mean and the error bars represent standard deviation of biological replicates. The error bars are hidden by the data points in this plot.
Activity remaining after a 20 min incubation at various temperatures is shown for the parent enzyme NphB M31 and the new enzyme NphB M31s. The data points reflect the mean and the error bars represent standard deviation of biological replicates.
Extended Data Fig. 5 Thermal Inactivation of MdcA from Pseudomonas putida Geobacillus stearothermophilus.
Activity remaining after a 20 min incubation at various temperatures is shown. The solvent conditions were 0.45 mg/ml P. putida MdcA (black) or 0.37 mg/ml enzyme G. stearothermophlus MdcA (blue) in 50 mM Tris [pH 8.0], 150 mM NaCl, 250 mM Imidizole, 30% glycerol. The data points reflect the mean and the error bars represent standard deviation of biological replicates.
We chose to proceed with 50 mM initial AcP because increasing the AcP concentration over 50 mM decreases the CBGA titer. The data points reflect the mean and the error bars represent standard deviation of biological replicates.
BSA titration data showing 20 mg/mL BSA should be used in subsequent reactions because there was minimal improvement when BSA was increased to 40 mg/mL. The data points reflect the mean and the error bars represent standard deviation of biological replicates.
Varying starting Acetate or Phosphate concentration from 0 to 100 mM had minimal effect on CBGA production using isoprenol and OA as inputs. The data points reflect the mean and the error bars represent standard deviation of biological replicates.
About this article
Cite this article
Valliere, M.A., Korman, T.P., Arbing, M.A. et al. A bio-inspired cell-free system for cannabinoid production from inexpensive inputs. Nat Chem Biol 16, 1427–1433 (2020). https://doi.org/10.1038/s41589-020-0631-9