A transient expression platform for rapid metabolic engineering of artemisinin biosynthesis in Artemisia annua
- 1Department of Biotechnology, University of Verona
- 2PlantaRei Biotech Srl
Abstract
Artemisinin, a sesquiterpene lactone produced in glandular trichomes of Artemisia annua , remains a key compound for artemisinin-based combination therapies against malaria. Although stable transformation has been widely used to enhance artemisinin production, rapid in planta systems for testing metabolic engineering strategies in A. annua remain limited. In this study, we developed a transient expression system based on vacuum agroinfiltration of whole A. annua plants, providing a fast and flexible tool to explore the engineering potential of the artemisinin pathway. Using GFP as a reporter, we established experimental conditions that support reproducible transient expression, identifying in vitro -grown plants as the most suitable material. We then assembled five key genes of the artemisinin biosynthetic pathway into a single multigene construct and tested its ability to redirect metabolism in both Nicotiana benthamiana and A. annua . While heterologous expression in N. benthamiana did not lead to detectable artemisinin-related metabolites, transient expression in A. annua produced a measurable, although temporary, shift in the specialized metabolome. At 2 dpi, targeted LC–HRMS analysis showed that artemisinin content increased from 0.086 ± 0.015 to 0.123 ± 0.012 mg/g fresh weight, while deoxyartemisinin and several putative sesquiterpenoid compounds also increased in relative abundance, suggesting that the introduced pathway genes influenced metabolic flux. These effects faded over time, revealing the strong endogenous regulatory constraints that shape artemisinin accumulation in its native plant host. Overall, this work establishes a rapid in planta platform for probing and modulating specialized metabolism in A. annua . Beyond artemisinin, it highlights the value of transient expression as a bridge between molecular design and plant-based production, supporting the broader vision of plants as dynamic biofactories for health-relevant natural products.
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BibTeX
@article{pietrolucci2026transient,
title = {{A transient expression platform for rapid metabolic engineering of artemisinin biosynthesis in Artemisia annua}},
author = {Fabio Pietrolucci and Vittoria Valesi and Elena Sgaravatti and Flavia Guzzo and Stefano Negri and Linda Avesani},
journal = {Frontiers in Plant Science},
year = {2026},
month = oct,
doi = {10.3389/fpls.2026.1876067},
url = {https://www.frontiersin.org/articles/10.3389/fpls.2026.1876067/full},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - A transient expression platform for rapid metabolic engineering of artemisinin biosynthesis in Artemisia annua
AU - Fabio Pietrolucci
AU - Vittoria Valesi
AU - Elena Sgaravatti
AU - Flavia Guzzo
AU - Stefano Negri
AU - Linda Avesani
PY - 2026
DA - 2026/10/21
JO - Frontiers in Plant Science
DO - 10.3389/fpls.2026.1876067
UR - https://www.frontiersin.org/articles/10.3389/fpls.2026.1876067/full
AB - Artemisinin, a sesquiterpene lactone produced in glandular trichomes of Artemisia annua , remains a key compound for artemisinin-based combination therapies against malaria. Although stable transformation has been widely used to enhance artemisinin production, rapid in planta systems for testing metabolic engineering strategies in A. annua remain limited. In this study, we developed a transient expression system based on vacuum agroinfiltration of whole A. annua plants, providing a fast and flexible tool to explore the engineering potential of the artemisinin pathway. Using GFP as a reporter, we established experimental conditions that support reproducible transient expression, identifying in vitro -grown plants as the most suitable material. We then assembled five key genes of the artemisinin biosynthetic pathway into a single multigene construct and tested its ability to redirect metabolism in both Nicotiana benthamiana and A. annua . While heterologous expression in N. benthamiana did not lead to detectable artemisinin-related metabolites, transient expression in A. annua produced a measurable, although temporary, shift in the specialized metabolome. At 2 dpi, targeted LC–HRMS analysis showed that artemisinin content increased from 0.086 ± 0.015 to 0.123 ± 0.012 mg/g fresh weight, while deoxyartemisinin and several putative sesquiterpenoid compounds also increased in relative abundance, suggesting that the introduced pathway genes influenced metabolic flux. These effects faded over time, revealing the strong endogenous regulatory constraints that shape artemisinin accumulation in its native plant host. Overall, this work establishes a rapid in planta platform for probing and modulating specialized metabolism in A. annua . Beyond artemisinin, it highlights the value of transient expression as a bridge between molecular design and plant-based production, supporting the broader vision of plants as dynamic biofactories for health-relevant natural products.
SN - 1664-462X
ER -