Presentation Information
[3GteX-15]Engineering rapid production of valuable triterpenoids in Nicotiana benthamiana leaves using a transient expression system
○Much Z. Fanani1, Kenji Miura2,3, Hikaru Seki1,4 (1. Grad. Sch. Eng., Univ. Osaka (Japan), 2. Bio. Sci., Univ. Tsukuba (Japan), 3. T-PIRC, Univ. Tsukuba (Japan), 4. OTRI, Univ. Osaka (Japan))
Keywords:
Plant biomanufacturing,Triterpenoids,Transient expression,Agroinfiltration,Nicotiana benthamiana,Plant specialized metabolites
[Purpose]
Establishing rapid and scalable platforms for producing complex plant metabolites remains a major challenge in biomanufacturing. While plants offer a promising and sustainable solution, efficient production systems are still limited, particularly for structurally complex compounds such as triterpenoids. Triterpenoids comprise a diverse class of high-value compounds, including bioactive saponins such as glycyrrhizin and glycyrrhetinic acid derivatives; however, their production still relies largely on extraction from natural plant sources, requiring long cultivation periods and raising sustainability concerns. To address these challenges, we developed a rapid and scalable platform for triterpenoid production using transient expression in N. benthamiana (tobacco) leaves.
[Method]
We used the Tsukuba system, an agroinfiltration-based transient expression platform with a geminiviral replicon and a double terminator-containing binary vector for high-level protein production in plants. Biosynthetic genes were individually cloned into the Tsukuba vector and introduced into Agrobacterium tumefaciens. Multiple strains carrying different pathway genes were combined and co-infiltrated into the leaves of 5–6-week-old tobacco plants. Leaves were harvested 5–7 days after infiltration, followed by metabolite extraction and analysis using LC–MS and GC–MS. To enhance metabolic flux, we combined pathway reconstruction (“pull”) with precursor supply engineering (“push”) using a feedback-insensitive truncated HMGR (tAtHMG1-S577A).
[Results]
We reconstructed triterpenoid biosynthetic pathways in tobacco through simultaneous expression of multiple enzymes, including oxidosqualene cyclase, cytochrome P450 monooxygenases, and glycosyltransferases, enabling production of glycyrrhizin and related triterpenoids. Implementation of a push strategy significantly enhanced metabolic flux, resulting in a 4.5-fold increase in glycyrrhizin accumulation to 254.16 ± 86.2 µg/g DW. Further optimization of Agrobacterium density (OD600 = 1.0) increased the yield to a maximum of 592.59 µg/g DW. Importantly, these structurally complex metabolites were produced within 5–7 days after agroinfiltration, representing a substantial acceleration compared to conventional plant-based production systems requiring months to years. These results demonstrate that transient expression enables rapid production and reconstitution of diverse triterpenoid pathways in planta.
[Consideration]
A key advantage of this platform is the ability to introduce multiple genes simultaneously by mixing Agrobacterium strains, enabling rapid assembly and optimization of complex biosynthetic pathways. Future improvements will require further engineering of host metabolism, including enhancement of precursor supply, reduction of competing pathways, and development of optimized tobacco chassis lines.
[Conclusion]
We established a plant-based platform for rapid production of valuable triterpenoids in tobacco via transient expression. This approach provides a fast, flexible, and scalable alternative to conventional plant extraction and represents a promising strategy for sustainable biomanufacturing of structurally complex triterpenoids, including compounds that are rare or difficult to obtain from natural sources.
[Acknowledgement]
This work was supported by the JST GteX Program, Japan, Grant Number JPMJGX23B0.
Establishing rapid and scalable platforms for producing complex plant metabolites remains a major challenge in biomanufacturing. While plants offer a promising and sustainable solution, efficient production systems are still limited, particularly for structurally complex compounds such as triterpenoids. Triterpenoids comprise a diverse class of high-value compounds, including bioactive saponins such as glycyrrhizin and glycyrrhetinic acid derivatives; however, their production still relies largely on extraction from natural plant sources, requiring long cultivation periods and raising sustainability concerns. To address these challenges, we developed a rapid and scalable platform for triterpenoid production using transient expression in N. benthamiana (tobacco) leaves.
[Method]
We used the Tsukuba system, an agroinfiltration-based transient expression platform with a geminiviral replicon and a double terminator-containing binary vector for high-level protein production in plants. Biosynthetic genes were individually cloned into the Tsukuba vector and introduced into Agrobacterium tumefaciens. Multiple strains carrying different pathway genes were combined and co-infiltrated into the leaves of 5–6-week-old tobacco plants. Leaves were harvested 5–7 days after infiltration, followed by metabolite extraction and analysis using LC–MS and GC–MS. To enhance metabolic flux, we combined pathway reconstruction (“pull”) with precursor supply engineering (“push”) using a feedback-insensitive truncated HMGR (tAtHMG1-S577A).
[Results]
We reconstructed triterpenoid biosynthetic pathways in tobacco through simultaneous expression of multiple enzymes, including oxidosqualene cyclase, cytochrome P450 monooxygenases, and glycosyltransferases, enabling production of glycyrrhizin and related triterpenoids. Implementation of a push strategy significantly enhanced metabolic flux, resulting in a 4.5-fold increase in glycyrrhizin accumulation to 254.16 ± 86.2 µg/g DW. Further optimization of Agrobacterium density (OD600 = 1.0) increased the yield to a maximum of 592.59 µg/g DW. Importantly, these structurally complex metabolites were produced within 5–7 days after agroinfiltration, representing a substantial acceleration compared to conventional plant-based production systems requiring months to years. These results demonstrate that transient expression enables rapid production and reconstitution of diverse triterpenoid pathways in planta.
[Consideration]
A key advantage of this platform is the ability to introduce multiple genes simultaneously by mixing Agrobacterium strains, enabling rapid assembly and optimization of complex biosynthetic pathways. Future improvements will require further engineering of host metabolism, including enhancement of precursor supply, reduction of competing pathways, and development of optimized tobacco chassis lines.
[Conclusion]
We established a plant-based platform for rapid production of valuable triterpenoids in tobacco via transient expression. This approach provides a fast, flexible, and scalable alternative to conventional plant extraction and represents a promising strategy for sustainable biomanufacturing of structurally complex triterpenoids, including compounds that are rare or difficult to obtain from natural sources.
[Acknowledgement]
This work was supported by the JST GteX Program, Japan, Grant Number JPMJGX23B0.
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