Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa
Terpenoids are the largest and most diverse family of natural products. Volatile terpenes from Cannabis sativa are crucial in flavours, fragrances, and pharmaceuticals due to their unique odours and biological activities, including antimalarial, antibacterial, and insecticidal properties. Their synt...
Saved in:
| Main Authors: | , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
PeerJ Inc.
2025-07-01
|
| Series: | PeerJ |
| Subjects: | |
| Online Access: | https://peerj.com/articles/19723.pdf |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849320910702510080 |
|---|---|
| author | Danielle Wiles James Roest Bhuvana Shanbhag Julian Vivian Travis Beddoe |
| author_facet | Danielle Wiles James Roest Bhuvana Shanbhag Julian Vivian Travis Beddoe |
| author_sort | Danielle Wiles |
| collection | DOAJ |
| description | Terpenoids are the largest and most diverse family of natural products. Volatile terpenes from Cannabis sativa are crucial in flavours, fragrances, and pharmaceuticals due to their unique odours and biological activities, including antimalarial, antibacterial, and insecticidal properties. Their synthesis is catalysed by terpene synthase (TPS) enzymes, which perform cyclisation and rearrangement reactions of over 55,000 distinct terpene compounds. However, low catalytic efficiency of C. sativa TPSs limits their use in large-scale commercial production. The complex biochemistry of these reactions is not well understood due to limited enzyme structure information. To address this, we have developed an integrated platform for the systematic expression, purification, enzymatic characterisation, and crystallisation of TPS enzymes from C. sativa. This workflow combines kinetic, thermostability, and structural analyses with a data-mining-informed crystallisation screen that enabled the production of diffraction-quality crystals. As a demonstration of the platform’s utility, ten C. sativa TPS enzymes were functionally characterised, revealing turnover rates (kcat) ranging from 0.0011 to 0.0204 s−1 and diverse substrate specificities, with each enzyme producing a distinct product profile, highlighting the need for systematic characterisation of C. sativa terpene biosynthesis. Our findings provide a framework for the structural and functional study of C. sativa TPSs. The developed platform sets the stage for future metabolic engineering aimed at optimising terpene production for pharmaceutical, pest management, and synthetic biology applications. |
| format | Article |
| id | doaj-art-023a4d56db0742fcb30eee8ed9b41c3a |
| institution | Kabale University |
| issn | 2167-8359 |
| language | English |
| publishDate | 2025-07-01 |
| publisher | PeerJ Inc. |
| record_format | Article |
| series | PeerJ |
| spelling | doaj-art-023a4d56db0742fcb30eee8ed9b41c3a2025-08-20T03:49:55ZengPeerJ Inc.PeerJ2167-83592025-07-0113e1972310.7717/peerj.19723Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativaDanielle Wiles0James Roest1Bhuvana Shanbhag2Julian Vivian3Travis Beddoe4Department of Ecological, Plant and Animal Science, La Trobe University, Bundoora, Victoria, AustraliaSt Vincent’s Institute of Medical Research, Fitzroy, Victoria, AustraliaDepartment of Ecological, Plant and Animal Science, La Trobe University, Bundoora, Victoria, AustraliaSt Vincent’s Institute of Medical Research, Fitzroy, Victoria, AustraliaDepartment of Ecological, Plant and Animal Science, La Trobe University, Bundoora, Victoria, AustraliaTerpenoids are the largest and most diverse family of natural products. Volatile terpenes from Cannabis sativa are crucial in flavours, fragrances, and pharmaceuticals due to their unique odours and biological activities, including antimalarial, antibacterial, and insecticidal properties. Their synthesis is catalysed by terpene synthase (TPS) enzymes, which perform cyclisation and rearrangement reactions of over 55,000 distinct terpene compounds. However, low catalytic efficiency of C. sativa TPSs limits their use in large-scale commercial production. The complex biochemistry of these reactions is not well understood due to limited enzyme structure information. To address this, we have developed an integrated platform for the systematic expression, purification, enzymatic characterisation, and crystallisation of TPS enzymes from C. sativa. This workflow combines kinetic, thermostability, and structural analyses with a data-mining-informed crystallisation screen that enabled the production of diffraction-quality crystals. As a demonstration of the platform’s utility, ten C. sativa TPS enzymes were functionally characterised, revealing turnover rates (kcat) ranging from 0.0011 to 0.0204 s−1 and diverse substrate specificities, with each enzyme producing a distinct product profile, highlighting the need for systematic characterisation of C. sativa terpene biosynthesis. Our findings provide a framework for the structural and functional study of C. sativa TPSs. The developed platform sets the stage for future metabolic engineering aimed at optimising terpene production for pharmaceutical, pest management, and synthetic biology applications.https://peerj.com/articles/19723.pdfCannabisTerpene synthaseStructureRecombinant expressionCrystallisation |
| spellingShingle | Danielle Wiles James Roest Bhuvana Shanbhag Julian Vivian Travis Beddoe Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa PeerJ Cannabis Terpene synthase Structure Recombinant expression Crystallisation |
| title | Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa |
| title_full | Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa |
| title_fullStr | Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa |
| title_full_unstemmed | Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa |
| title_short | Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa |
| title_sort | integrated platform for structural and functional analysis of terpene synthases of cannabis sativa |
| topic | Cannabis Terpene synthase Structure Recombinant expression Crystallisation |
| url | https://peerj.com/articles/19723.pdf |
| work_keys_str_mv | AT daniellewiles integratedplatformforstructuralandfunctionalanalysisofterpenesynthasesofcannabissativa AT jamesroest integratedplatformforstructuralandfunctionalanalysisofterpenesynthasesofcannabissativa AT bhuvanashanbhag integratedplatformforstructuralandfunctionalanalysisofterpenesynthasesofcannabissativa AT julianvivian integratedplatformforstructuralandfunctionalanalysisofterpenesynthasesofcannabissativa AT travisbeddoe integratedplatformforstructuralandfunctionalanalysisofterpenesynthasesofcannabissativa |