ORIGINAL RESEARCH article
Front. Plant Sci.
Sec. Plant Biotechnology
Volume 16 - 2025 | doi: 10.3389/fpls.2025.1678814
This article is part of the Research TopicPlant Molecular Farming for Biopharmaceutical Production and BeyondView all 10 articles
Efficient accumulation of new irregular monoterpene malonyl glucosides in Nicotiana benthamiana achieved by co-expression of isoprenyl diphosphate synthases and substrate-producing enzymes
Provisionally accepted
- Darmstadt University of Technology, Darmstadt, Germany
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Irregular monoterpenes have limited natural sources but possess unique activities applicable in medicine and agriculture. To enable sustainable plant-based production of these compounds, we established a transient expression procedure to enhance the biosynthetic flux in Nicotiana benthamiana toward dimethylallyl diphosphate (DMAPP), a substrate for isopentenyl diphosphate synthases (IDSs) that generate irregular monoterpene skeletons. Considering the benefits of glycosylation for accumulating and storing monoterpenes in extractable form, we focused on developing a platform for production of non-volatile glycosylated irregular monoterpenes using three IDS that form branched and cyclic structures. The analysis of methanolic leaf extracts from transiently transformed N. benthamiana plants revealed six major new components, 6-O-malonyl-β-D-glucopyranoside and 6-O-malonyl-β-D-glucopyranosyl-(1→2)-β-D-glucopyranoside derivatives of chrysanthemol, lavandulol and cyclolavandulol, five of which are novel compounds. Alleviating two bottlenecks in the DMAPP formation in plastids by co-expressing 1-deoxyxylulose 5-phosphate synthase and isopentenyl diphosphate isomerases increased the yield of chrysantemyl and lavandulyl glucosides produced by plant-derived IDS to 1.7 ± 0.4 μmol g-1 FW and 1.4 ± 0.3 μmol g-1 FW, respectively. A bacterial cyclolavandulyl diphosphate synthase operated efficiently in chloroplasts and cytoplasm. The highest irregular monoterpene concentrations were achieved in cytoplasm by co-expression of hydroxymethylglutaryl-CoA reductase, the bottleneck enzyme of the mevalonate pathway for DMAPP biosynthesis. The mean level of cyclolavandulyl glucosides reached 3.9 ± 1.5 μmol g-1 FW; the top-performing plants contained 6.6 μmol g-1 FW. This yield represents the highest amount of irregular monoterpene glycosides produced in plant systems.
Keywords: Irregular monoterpenes, Lavandulol, cyclolavandulol, Chrysanthemol, Nicotiana benthamiana, DMAPP biosynthetic pathway engineering
Received: 03 Aug 2025; Accepted: 15 Sep 2025.
Copyright: © 2025 Gerasymenko, Sheludko, Schmidts and Warzecha. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
* Correspondence: Iryna Gerasymenko, gerasymenko@bio.tu-darmstadt.de
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