Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization
The production of cannabinoid compounds such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) with potential pharmaceutical applications is growing sharply. However, challenges such as the low yield of minor cannabinoids, legal restrictions on cultivation, and the comple...
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Elsevier
2025-03-01
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| Series: | Biotechnology Reports |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2215017X24000389 |
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| author | Omayra C. Bolaños-Martínez Anna Urbanetz Daniel Maresch Richard Strasser Sornkanok Vimolmangkang |
| author_facet | Omayra C. Bolaños-Martínez Anna Urbanetz Daniel Maresch Richard Strasser Sornkanok Vimolmangkang |
| author_sort | Omayra C. Bolaños-Martínez |
| collection | DOAJ |
| description | The production of cannabinoid compounds such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) with potential pharmaceutical applications is growing sharply. However, challenges such as the low yield of minor cannabinoids, legal restrictions on cultivation, and the complexity and cost of purification from the Cannabis sativa plant necessitate a biotechnological approach. Since the biosynthetic pathway is disclosed, cannabinoids have been produced in yeast, insect cells and plants mainly by the heterologous expression of tetrahydrocannabinol acid synthase (THCAS). THCAS and cannabidiolic acid synthase (CBDAS) use cannabigerolic acid (CBGA) as a substrate. In this study, we transiently expressed recombinant forms of THCAS and CBDAS in leaves of Nicotiana benthamiana. Our results demonstrate that efficient expression in the secretory pathway relies on replacing the endogenous signal peptide with a heterologous one. Both proteins were successfully secreted to the apoplast. MS-based analysis of the purified proteins revealed that they are heavily glycosylated with mainly Golgi-processed complex type N-glycans. In planta enzymatic removal of N-glycans indicated that glycosylation plays a role for CBDAS protein folding or stability. Finally, in vitro assays with CBGA showed that the plant-made recombinant CBDAS and THCAS are enzymatically active. |
| format | Article |
| id | doaj-art-dba8ef821eb54232a47426674b6a18f1 |
| institution | OA Journals |
| issn | 2215-017X |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Biotechnology Reports |
| spelling | doaj-art-dba8ef821eb54232a47426674b6a18f12025-08-20T02:00:46ZengElsevierBiotechnology Reports2215-017X2025-03-0145e0086510.1016/j.btre.2024.e00865Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimizationOmayra C. Bolaños-Martínez0Anna Urbanetz1Daniel Maresch2Richard Strasser3Sornkanok Vimolmangkang4Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; Center of Excellence in Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok 10330, Thailand; Department of Applied Genetics and Cell Biology, Institute of Plant Biotechnology and Cell Biology, University of Natural Resources and Life Sciences, 1190 Vienna, AustriaCore Facility Mass Spectrometry, University of Natural Resources and Life Sciences, Vienna, AustriaCore Facility Mass Spectrometry, University of Natural Resources and Life Sciences, Vienna, AustriaDepartment of Applied Genetics and Cell Biology, Institute of Plant Biotechnology and Cell Biology, University of Natural Resources and Life Sciences, 1190 Vienna, AustriaDepartment of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; Center of Excellence in Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok 10330, Thailand; Corresponding author.The production of cannabinoid compounds such as Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD) and cannabichromene (CBC) with potential pharmaceutical applications is growing sharply. However, challenges such as the low yield of minor cannabinoids, legal restrictions on cultivation, and the complexity and cost of purification from the Cannabis sativa plant necessitate a biotechnological approach. Since the biosynthetic pathway is disclosed, cannabinoids have been produced in yeast, insect cells and plants mainly by the heterologous expression of tetrahydrocannabinol acid synthase (THCAS). THCAS and cannabidiolic acid synthase (CBDAS) use cannabigerolic acid (CBGA) as a substrate. In this study, we transiently expressed recombinant forms of THCAS and CBDAS in leaves of Nicotiana benthamiana. Our results demonstrate that efficient expression in the secretory pathway relies on replacing the endogenous signal peptide with a heterologous one. Both proteins were successfully secreted to the apoplast. MS-based analysis of the purified proteins revealed that they are heavily glycosylated with mainly Golgi-processed complex type N-glycans. In planta enzymatic removal of N-glycans indicated that glycosylation plays a role for CBDAS protein folding or stability. Finally, in vitro assays with CBGA showed that the plant-made recombinant CBDAS and THCAS are enzymatically active.http://www.sciencedirect.com/science/article/pii/S2215017X24000389CannabinoidsPlant biotechnologyHeterologous expressionApoplastic fluidComplex N-glycans |
| spellingShingle | Omayra C. Bolaños-Martínez Anna Urbanetz Daniel Maresch Richard Strasser Sornkanok Vimolmangkang Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization Biotechnology Reports Cannabinoids Plant biotechnology Heterologous expression Apoplastic fluid Complex N-glycans |
| title | Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization |
| title_full | Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization |
| title_fullStr | Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization |
| title_full_unstemmed | Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization |
| title_short | Engineering Nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization |
| title_sort | engineering nicotiana benthamiana for production of active cannabinoid synthases via secretory pathway optimization |
| topic | Cannabinoids Plant biotechnology Heterologous expression Apoplastic fluid Complex N-glycans |
| url | http://www.sciencedirect.com/science/article/pii/S2215017X24000389 |
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