Bibenzyl synthesis in Cannabis sativa L.
Open accessAbstract
SUMMARY: This study focuses on the biosynthesis of a suite of specialized metabolites from Cannabis that are known as the ‘bibenzyls’. In planta , bibenzyls accumulate in response to fungal infection and various other biotic stressors; however, it is their widely recognized anti‐inflammatory properties in various animal cell models that have garnered recent therapeutic interest. We propose that these compounds are synthesized via a branch point from the core phenylpropanoid pathway in Cannabis , in a three‐step sequence. First, various hydroxycinnamic acids are esterified to acyl‐coenzyme A (CoA) by a member of the 4‐coumarate‐CoA ligase family (Cs4CL4). Next, these CoA esters are reduced by two double‐bond reductases (CsDBR2 and CsDBR3) that form their corresponding dihydro‐CoA derivatives from preferred substrates. Finally, the bibenzyl backbone is completed by a polyketide synthase that specifically condenses malonyl‐CoA with these dihydro‐hydroxycinnamoyl‐CoA derivatives to form two bibenzyl scaffolds: dihydropiceatannol and dihydroresveratrol. Structural determination of this ‘bibenzyl synthase’ enzyme (CsBBS2) indicates that a narrowing of the hydrophobic pocket surrounding the active site evolved to sterically favor the non‐canonical and more flexible dihydro‐hydroxycinnamoyl‐CoA substrates in comparison with their oxidized relatives. Accordingly, three point mutations that were introduced into CsBBS2 proved sufficient to restore some enzymatic activity with an oxidized substrate, in vitro . Together, the identification of this set of Cannabis enzymes provides a valuable contribution to the growing ‘parts prospecting’ inventory that supports the rational metabolic engineering of natural product therapeutics.
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STS and BBS accommodate the same precursors, namely cinnamic acid thioesters, particularly p-coumaric and caffeic acids for STS and their dihydrated equivalents for BBS.
“These two enzymes essentially accommodate the same precursors, namely cinnamic acid thioesters, particularly p-coumaric and caffeic acids for STS and their dihydrated equivalents for BBS [62].”
Ferulic acid and cinnamic acid (and their dihydrated homologues) can be accommodated by these enzymes, but significantly less well than their preferred substrates, while sinapic acid is not viable at all as a precursor.
“It is useful to note that ferulic acid and cinnamic acid (and their dihydrated homologues) can be accommodated by these enzymes, but significantly less well than their preferred substrates, while sinapic acid is not viable at all as a precursor for these enzymes [62].”
Boddington et al. identified nine C. sativa double bond reductase homologues (CsDBR1-9) in Cannabis, all classified as NAD(P)H-dependent alkenal/one reductases.
“Boddington et al. identified nine C. sativa double bond reductase homologues (CsDBR1-9) in Cannabis, and all are classified as NAD(P)H-dependent alkenal/one reductases.”
CsDBR2 and CsDBR3 catalyze the reduction of p-coumaroyl-CoA to dihydro-p-coumaroyl-CoA and caffeoyl-CoA to dihydrocaffeoyl-CoA, respectively.
“They determined that the enzymes CsDBR2 and CsDBR3 catalyze the reduction reaction of p-coumaroyl-CoA to dihydro-p-coumaroyl-CoA, and caffeoyl-CoA to dihydrocaffeoyl-CoA, respectively.”
A structural study of CsBBS2 indicates it has a narrowing at the hydrophobic pocket of the active site compared to STS, explaining its greater affinity for the dihydrated form of cinnamic acid thioesters.
“A structural study of the Cannabis bibenzyl synthase 2 enzyme (CsBBS2) indicates that it has undergone a narrowing at the level of the hydrophobic pocket of the active site, when compared to STS, explaining the greater affinity for the dihydrated form of cinnamic acid thioesters [62].”
Enzymatic tests indicate CsBBS2 accommodates dihydro-p-coumaroyl-CoA and dihydrocaffeoyl-CoA, while CsBBS1 is incapable of doing so.
“Their results of enzymatic tests on the enzymes CsBBS1 and CsBBS2, suspected of operating the biosynthesis of bibenzyls in Cannabis, indicate that the enzyme CsBBS2 accommodates the two substrates, dihydro-p-coumaroyl-CoA and dihydrocaffeoyl-CoA, while CsBBS1 is incapable of doing so [62].”
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Biosynthesis of cannflavins A and B from Cannabis sativa L.
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Therapeutic Potential and Predictive Pharmaceutical Modeling of Stilbenes in Cannabis sativa
O’Croinin, C., Garcia Guerra, A., Doschak, M. R., Löbenberg, R., & Davies, N. M. (2023) · Pharmaceutics · doi:10.3390/pharmaceutics15071941 · Cited by 25 · see citing works
Boucher, R., Germain, H., & Desgagné-Penix, I. (2025) · Plants · doi:10.3390/plants14091372 · Cited by 23 · see citing works
- Authors
- Boddington, K. F., Soubeyrand, E., Van Gelder, K., Casaretto, J. A., Perrin, C., Forrester, T. J. B., Parry, C., Al‐Abdul‐Wahid, M. S., Jentsch, N. G., Magolan, J., Bozzo, G. G., Kimber, M. S., Rothstein, S. J., & Akhtar, T. A.
- Affiliations
- University of Guelph (CA)
- Department of Molecular and Cellular Biology University of Guelph Guelph Ontario N1G 2W1 Canada
- NMR Centre University of Guelph Guelph Ontario N1G 2W1 Canada
- McMaster University (CA)
- Corresponding author
- Akhtar, Tariq A.
- ORCID
- Venue
- The Plant Journal
- Publisher
- Wiley
- Published
- 2021-12-06
- Volume
- 109
- Issue
- 3
- Pages
- 693-707
- ISSN
- 0960-7412 (ISSN-L)
- 1365-313X
- OpenAlex
- W3213831250
- Cited by
- 21
- References
- 2
- Citations by year
- 2026: 6 · 2025: 3 · 2024: 7 · 2023: 1 · 2022: 4
- FWCI
- 0.94
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- The Plant Journal (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant biochemistry and biosynthesis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Plant Gene Expression Analysis
- Health Sciences › Medicine › Pharmacology › Microbial Natural Products and Biosynthesis
- Keywords
- bibenzyls, Cannabis sativa, specialized metabolite biosynthesis, phenylpropanoid pathway, 4-coumarate:CoA ligase, polyketide synthase, fungal infection response, anti-inflammatory agents, site-directed mutagenesis, metabolic engineering
- MeSH terms
- Anti-Inflammatory Agents, Bibenzyls, Cannabis, Plants, Medicinal, Biosynthetic Pathways
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