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A bio-inspired cell-free system for cannabinoid production from inexpensive inputs


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.

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Fig. 1: Cannabinoid biosynthesis.
Fig. 2: Cell-free system design for cannabinoid production.
Fig. 3: Testing the AP module and MatB system.
Fig. 4: System testing.
Fig. 5: Implementation of the full cannabinoid production system.

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This work was supported by Department of Energy grant nos. DE-FC02-02ER63421 and DE-AR0000556 to J.U.B.

Author information

Authors and Affiliations



M.A.V. developed system designs in consultation with T.P.K. and J.U.B. All authors contributed to experimental design and data analysis, but M.A.V. designed the bulk of the experiments. M.A.V. also conducted the bulk of the experiments with assistance from T.P.K. and M.A.A. M.A.V. and J.U.B. wrote the manuscript with contributions from T.P.K. and M.A.A.

Corresponding author

Correspondence to James U. Bowie.

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Competing interests

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.

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Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 The effect of OLS and AAE3 concentrations on product specificity.

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.

Extended Data Fig. 2 OA inhibition of enzyme activity.

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.

Extended Data Fig. 3 Inhibition of OA and CBGA production by GPP.

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.

Extended Data Fig. 4 Stabilization of NphB M31.

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.

Extended Data Fig. 6 The titer of CBGA as a function of initial AcP concentrations.

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.

Extended Data Fig. 7 The effect of BSA on the titer of OA using MdcA to generate malonyl-CoA.

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.

Extended Data Fig. 8 The effect of acetate and phosphate on CBGA production.

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.

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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).

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