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Parallel evolution of cannabinoid biosynthesis

Nature Plants volume 9, pages 817–831 (2023)Cite this article

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Abstract

Modulation of the endocannabinoid system is projected to have therapeutic potential in almost all human diseases. Accordingly, the high demand for novel cannabinoids stimulates the discovery of untapped sources and efficient manufacturing technologies. Here we explored Helichrysum umbraculigerum, an Asteraceae species unrelated to Cannabis sativa that produces Cannabis-type cannabinoids (for example, 4.3% cannabigerolic acid). In contrast to Cannabis, cannabinoids in H. umbraculigerum accumulate in leaves’ glandular trichomes rather than in flowers. The integration of de novo whole-genome sequencing data with unambiguous chemical structure annotation, enzymatic assays and pathway reconstitution in Nicotiana benthamiana and in Saccharomyces cerevisiae has uncovered the molecular and chemical features of this plant. Apart from core biosynthetic enzymes, we reveal tailoring ones producing previously unknown cannabinoid metabolites. Orthology analyses demonstrate that cannabinoid synthesis evolved in parallel in H. umbraculigerum and Cannabis. Our discovery provides a currently unexploited source of cannabinoids and tools for engineering in heterologous hosts.

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Fig. 1: H. umbraculigerum biosynthesizes CBGA and other terpenophenols in all aerial plant parts.
Fig. 2: Cannabinoid-associated gene expression is correlated with cannabinoid metabolite accumulation in H. umbraculigerum glandular trichomes.
Fig. 3: Discovery of the core cannabinoid biosynthetic pathway enzymes.
Fig. 4: Functional characterization of cannabinoid tailoring enzymes.
Fig. 5: In vivo reconstruction of the core cannabinoid pathway in heterologous systems.
Fig. 6: Parallel and divergent evolution of the cannabinoid biosynthetic pathway.

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Data availability

All NGS raw sequencing data as well as the primary genome assembly version Humb_v1 can be found in ENA under the accession number PRJEB52026. The sequences of the active genes reported in this Article have been deposited in NCBI GenBank (accessions OQ673107–OQ673113). The genomic position, functional annotation, CDSs and protein sequences of these genes are available in Supplementary Tables 14 and 19. The protein databases used in this study are Uniprot Swiss-Prot release-2022_02, sunflower UP000215914_4232, Arabidopsis UP000006548_3702, tomato UP000004994_4081, rice UP000059680_39947 and Cannabis NCBI GCF_900626175.1_cs10. The Pfam database was Pfam-A.hmm release 34.0. Source data are provided with this paper.

Code availability

All the code used in this study along with a description of the scripts can be found at https://github.com/Luisitox/Helichrysum_paper.

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Acknowledgements

A.A. is the incumbent of the Peter J. Cohn Professorial Chair. L.A.d.H. was partially supported by fellowships from the Israel Ministry of Absorption and the Dean of the Plant Science Department in the Weizmann Institute. We thank XINTEZA (https://xinteza.com/) for funding this research. We thank the Adelis Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, the Jeanne and Joseph Nissim Foundation for Life Sciences, Tom and Sondra Rykoff Family Foundation Research, Ron Sklare and the Raymond Burton Plant Genome Research Fund for supporting the A.A. laboratory. T.S. is the incumbent of the Monroy-Marks Research Fellow Chair. We thank S. Arava for the help in the purification of metabolites and N. Shahaf for support in applying the WEIZMASS library of plant metabolites. The electron microscopy studies were conducted at the Irving and Cherna Moskowitz Center for Nano and Bio-Nano Imaging at the Weizmann Institute of Science.

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Author notes

  1. Prashant D. Sonawane

    Present address: Department of Natural Product Biosynthesis, Max-Planck Institute for Chemical Ecology, Jena, Germany

  2. These authors contributed equally: Paula Berman, Luis Alejandro de Haro, Adam Jozwiak.

Authors and Affiliations

  1. Department of Plant and Environmental Sciences, Weizmann Institute of Science, Rehovot, Israel

    Paula Berman, Luis Alejandro de Haro, Adam Jozwiak, Sayantan Panda, Zoe Pinkas, Younghui Dong, Jelena Cveticanin, Rotem Livne, Sagit Meir, Ilana Rogachev, Prashant D. Sonawane & Asaph Aharoni

  2. Biochemical Sciences Division, CSIR-National Chemical Laboratory, Pune, India

    Ranjit Barbole

  3. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, India

    Ranjit Barbole

  4. Department of Chemical Research Support, Weizmann Institute of Science, Rehovot, Israel

    Tali Scherf, Eyal Shimoni, Smadar Levin-Zaidman & Nili Dezorella

  5. Life Science Core Facilities, Weizmann Institute of Science, Rehovot, Israel

    Ekaterina Petrovich-Kopitman

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Contributions

P.B., L.A.d.H., A.J., P.D.S. and A.A. designed the study. P.B., L.A.d.H. and A.A. wrote the manuscript with the assistance and input of all coauthors. P.B. led the chemical profiling, feeding and MALDI imaging experiments with the aid of A.J., J.C., Y.D. and I.R. L.A.d.H. performed all the bioinformatic analyses. P.B., L.A.d.H., A.J., P.D.S., R.B. and R.L. cloned and performed the in vitro enzymatic assays. A.J. performed the confocal measurements and the heterologous expression of genes in S. cerevisiae. S.P. prepared the libraries for Transeq sequencing. S.L.-Z., E.S. and N.D. performed the electron microscopy studies. Z.P. performed the transient co-expression of genes in N. benthamiana. P.B. and S.L.-Z. analysed the electron microscopy data. T.S. acquired and analysed the NMR data. E.P.-K. performed the flow-cytometry-based genome size estimation. S.M. cultivated and propagated the plants. A.A. supervised the study. All the authors read and approved the final version of the manuscript.

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Correspondence to Paula Berman, Prashant D. Sonawane or Asaph Aharoni.

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Nature Plants thanks John D’Auria, Jonathan Page and Jing-Ke Weng for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 LC-MS/MS fingerprinting of CBGA 1, heliCBGA 2 and APHA 3 in H. umbraculigerum.

a. Extracted ion chromatograms and MS/MS spectral matching of cannabigerolic acid (CBGA 1 [M-H]- = 359.222 Da), heli-cannabigerolic acid (heliCBGA 2 [M-H]- = 393.206 Da), and pre-amorphastilbol (APHA 3 [M-H]- = 391.191 Da) standards or authentic metabolites versus a H. umbraculigerum leaf extract. To confirm the assignment, CBGA 1 and heliCBGA 2 were purified and analyzed by NMR (Supplementary NMR Data 1, 2). b. Stable isotope labeling of CBGA 1, heliCBGA 2 and APHA 3 via feeding of H. umbraculigerum leaves with hexanoic-D11 acid, phenylalanine-D5 or phenylalanine-13C9. The MS/MS spectra of the non-labeled versus the labeled forms show similar fragmentation patterns with mass shifts corresponding with the labeled parts of the molecule.

Extended Data Fig. 2 Stalked glandular trichomes in leaves and flowers of H. umbraculigerum.

a., b. Representative cryo-SEM micrographs of the lateral view of flowers showing stalked glandular trichomes (marked by arrows). Micrographs are representative of multiple (n > 3) flower areas sampled on the same day. c. Light micrograph showing the biseriate structure of stalked glandular trichomes of leaves (n > 3). d.-f. Selected TEM micrographs of leaves’ trichomes at different stages of secretion. Micrographs are representative of distinct trichomes (n = 2–5 for each developmental stage) from sections of young and old leaves. High magnification images show the ultrastructure of disk cells (DCs). CW, cell wall; M, mitochondria; N, nucleus; P, plastid; PSP, periplasmic space; SCv, secretory cavity; V, vacuole; Vs, vesicle. d. In the presecretory stage, DCs contained a very dense cytoplasm covered by ER and multiple ribosomes. There was no SCv or PSP and plastids were large and resembled pro-plastids. e. In the secretory stage, delamination of the apical DC wall led to the formation of the SCv. Electron transparent secretions were exuded out of plastids in vesicles delimited by an electron-dense layer. The vesicles released their contents to the PSP by exocytosis where the secretory product accumulated prior to secretion into the SCv (marked by arrow). f. DCs of mature trichomes post-secretion were largely vacuolated with a cytoplasm restricted to the small remaining area. Plastids at this stage had degenerated and no vesicles were observed. The cell wall had a largely cutinized layer with a large SCv. MALDI-MSI of m/z 361.23 ± 0.01 Da signals of the g. abaxial and h. adaxial leaf domains (n = 1), following partial removal of trichomes by duct tape (the peeled area is outlined by green line). The areas with partially/fully removed trichomes show less or no signals compared to the untouched parts. i. Optical image and j. MALDI-MSI of m/z 361.23 ± 0.01 Da of a cross-sectioned flower receptacle (n = 2). Glandular trichomes in i are marked to improve interpretation. The signals in g., h. and j. correspond with the protonated m/z of CBGA 1 and geranylphlorocaprophenone 4. The white broken lines in g.-j. mark the regions analyzed.

Extended Data Fig. 3 Predicted parallel metabolic pathways for the biosynthesis of cannabinoids and other terpenophenols present in H. umbraculigerum.

The predicted types of enzymes catalyzing each reaction are marked by 1–8. Additional functional groups and rearrangements include hydroxylation, double bond isomerization or reduction, cyclization and others. Alkyl chains can be linear/branched with one to seven carbons length; AAE, acyl activating enzyme; PKS, type III polyketide synthase; PKC, polyketide cyclase; PT, prenyl-transferase; UGT, uridine diphosphate-glycosyltransferase; AAT, alcohol acyl transferase; DBR, double bond reductase; CHI, chalcone isomerase. The active enzymes identified in this study are marked by their names. CoAT, acyl-CoA-transferase; TKS, tetraketide synthase; CBGAS, cannabigerolic acid synthase; OAGT, olivetolic acid UGT; CBGT, cannabinoid UGT; CBAT, cannabinoid acyl-transferase.

Extended Data Fig. 4 Functional characterization of HuAAE, HuPKS and HuPTs.

a, Ion abundances from triple-Quad analyses of acyl-CoAs produced in vitro by the HuAAEs versus analytical standard (Std). b. A scheme showing the steps and types of products and by-products synthesized in vitro by the recombinant HuPKSs with or without the Cannabis olivetolic acid cyclase (CsOAC). c. Ion abundances from triple-Quad analyses of OA 92 and olivetol products from coupled recombinant enzyme assays of HuPKSs with either an empty vector (EV) or Cannabis olivetolic acid cyclase (CsOAC), in the presence of hexanoyl-CoA and malonyl-CoA. d. MS/MS spectra of prenylated OA 92 products with cannabigerolic acid synthase (HuCBGAS4) and either isopentenyl pyrophosphate (IPP), geranyl pyrophosphate (GPP) or farnesyl pyrophosphate (FPP) as the prenyl donors. CBPA 19, cannabiprenylic acid; CBGA 1, cannabigerolic acid; sesquiCBGA, sesqui cannabigerolic acid (MS/MS spectrum corresponds to published data from Cannabis15). e. Steady state kinetic analysis of H. umbraculigerum prenyl-transferases HuPT1, HuPT3 and HuCBGAS4 with OA 92 and GPP. The Michaelis–Menten Km value of each enzyme was calculated using varying (0.5 μM–3 mM) and constant (1 mM) concentrations of each substrate (mean ± s.d.; n = 3 technically independent samples).

Extended Data Fig. 5 Phylogenetic analyses of enzymes and whole proteome from H. umbraculigerum and different plant species.

Phylogenetic analysis of a. acyl-activating enzyme (AAE), b. polyketide synthase (PKS) and c. prenyl transferase (PT) proteins from H. umbraculigerum and other plants. Full list of protein IDs is available in Supplementary Table 15. Bootstrap values are indicated at the nodes of each branch. The selection of the proteins was based on a. Arabidopsis thaliana enzymes44 or b.,c. functionally tested enzymes22,45. Clades according to substrates or functionalities are marked by different colors. None of the active H. umbraculigerum enzymes clustered with any of the known Cannabis proteins. d. Phylogenetic relationship between Arabidopsis thaliana, Solanum lycopersicum, Helianthus annuus, Lactuca sativa, Cannabis sativa and Helicrysum umbraculigerum illustrate the evolutionary distance between the last two species. The tree was constructed based on the whole proteomes of each species using the word-based software Prot-SpaM.

Extended Data Fig. 6 Functional characterization of HuUGTs.

a. Activities of lysates containing HuUGTs with OA 92, CBGA 1 and heliCBGA 2 as substrates and uridine diphosphate glucose (UDP-Glc) as the sugar donor (n = 1). Reactions show differing substrate specificities and type of products. Representative peaks correspond to chromatograms obtained for HuCBUGT1. EV, empty vector. b. In vitro production of monoglucosides with the purified UGTs and additional substrates. Extracted ion chromatograms of the observed monoglucosides using UDP-Glc and either DHSA 93, olivetol, CBG, CBD, Δ9-THC, PCP 95, naringenin chalcone 97 or pinocembrin chalcone 100. The substrates naringenin chalcone 97 and pinocembrin chalcone 100 contained mixtures of the chalcones and respective flavanones. All LC-MS chromatograms were selected for the theoretical m/z values of the respective metabolites of interest. c. Comparison of steady state kinetics of UGTs with OA 92 and UDP-Glc. HuOAUGT11 and HuUGT13 were compared with UGTs from rice (OsUGT) and stevia (SrUGT). Kinetic values were calculated using varying (0.5 μM–3 mM) and constant (1 mM) concentrations of each substrate (mean ± s.d.; n = 3 technically independent samples; measurements were plotted individually). V0 and Vmax were calculated using the calibration curve of OA 92 since there was no analytical standard available for Glc-OA 102.

Extended Data Fig. 7 Functional characterization of HuAATs.

a. Stable dual isotope labeling of O-MeButCBGA 120 via feeding of H. umbraculigerum leaves with either 2-methyl butyric-D9 acid or hexanoic-D11 acid. The MS/MS spectra of the non-labeled versus the two-labeled forms show fragmentation patterns with mass shifts corresponding with the labeled parts of the molecule. b. Activities of lysates containing HuAATs with different acyl donors and cannabinoid acceptors. Extracted ion chromatograms were selected for the theoretical m/z values of the respective metabolites. Only HuCBAT5 and HuAAT14 (red and blue, respectively) acylated CBGA 1 and heliCBGA 2 with both acyl-CoAs. EV, empty vector; Std, standard; ButCoA, butyryl-CoA; HexCoA, hexanoyl-CoA. c. Phylogenetic analysis of HuAAT proteins and identified BAHD AATs from other plants. The Maximum Likelihood tree was constructed with 100 bootstrap tests based on a MUSCLE multiple alignment using the MEGA11 software. The evolutionary distances were computed using the JTTmatrix-based method. Bootstrap values are indicated at the nodes of each branch. The clades of the different AAT types are marked in circles based on Tuominen et al.26. The active HuCBAT5 and HuAAT14 were clustered in clade IIIa which represents BAHDs of diverse catalytic functions. Full list of protein IDs is available in Supplementary Table 15.

Extended Data Fig. 8 MS/MS spectra of observed O-acylated cannabinoids following enzymatic assays with the purified HuCBAT5.

OA 92, olivetolic acid; CBGA 1, cannabigerolic acid; HeliCBGA 2, helicannabigerolic acid; CBDA, cannabidiolic acid. Full data of MS/MS products appears in Supplementary Table 21. MS/MS fragmentation and retention times correspond to the O-acylated cannabinoids found in the plant.

Extended Data Fig. 9 Reconstruction of the core cannabinoid pathway in heterologous systems.

Schematic representation of products observed in a. N. benthamiana leaves and d. S. cerevisiae yeasts following co-expression of different combinations of HuCoAT6, HuTKS4, and HuCBGAS4, along with CsOAC from Cannabis. NbUGT, putative N. benthamiana uridine diphosphate-glycosyltransferase; HexNa, sodium hexanoate; GPP, geranyl pyrophosphate; OA 92, olivetolic acid. Extracted ion chromatograms and MS/MS spectra showing b. glycosylated OA (Glc-OA 102), glycosylated polycaprophenone (Glc-PCP1/2) and glycosylated naringenin chalcone (Glc-Naringenin chalcone 1/2) following feeding with HexNa and GPP (I); and c. glycosylated cannabigerolic acid (Glc-CBGA 109) following feeding with OA 92 and GPP (II). Glycosylated metabolites synthesized by the recombinant stevia (SrUGT) or rice (OsUGT) enzymes were used as reference for identification of N. benthamiana products according to exact mass, retention time and MS/MS spectra. EV, empty vector; UDP-Glc, uridine diphosphate glucose. e. Extracted ion chromatograms of OA 92, PCP 95 and CBGA 1 products observed in yeasts without any feeding. Identification was according to analytical standards. f. Summary of the observed products in each assay. PDAL, pentyl acyl diacetic acid lactone; HTAL, hexanoyl acyl triacetic acid lactone.

Supplementary information

Supplementary Information

Supplementary Figs. 1–26, NMR Data 1–14, Orthology Data 1–4, Methods and table captions.

Reporting Summary

Supplementary Tables

Supplementary Tables 1–23.

Source data

Source Data Fig. 1

CBGA content in different tissues.

Source Data Fig. 3

Statistical source data.

Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Statistical source data.

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Berman, P., de Haro, L.A., Jozwiak, A. et al. Parallel evolution of cannabinoid biosynthesis. Nat. Plants 9, 817–831 (2023). https://doi.org/10.1038/s41477-023-01402-3

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