Volatile science? Metabolic engineering of terpenoids in plants Asaph Aharoni 1 , Maarten A. Jongsma 2 and Harro J. Bouwmeester 2 1 Weizmann Institute of Science, PO Box 26, Rehovot 76100, Israel 2 Plant Research International, PO Box 16, 6700 AA Wageningen, The Netherlands Terpenoids are important for plant survival and also possess biological properties that are beneficial to humans. Here, we describe the state of the art in terpenoid metabolic engineering, showing that signifi- cant progress has been made over the past few years. Subcellular targeting of enzymes has demonstrated that terpenoid precursors in subcellular compartments are not as strictly separated as previously thought and that multistep pathway engineering is feasible, even across cell compartments. These engineered plants show that insect behavior is influenced by terpenoids. In the future, we expect rapid progress in the engineering of terpenoid production in plants. In addition to commercial appli- cations, such transgenic plants should increase our understanding of the biological relevance of these volatile secondary metabolites. Importance of terpenoids Isoprenoids, also known as terpenoids, are the largest family of natural compounds, consisting of O40 000 different molecules. The isoprenoid biosynthetic pathway generates both primary and secondary metabolites that are of great importance to plant growth and survival. Among the primary metabolites produced by this pathway are: the phytohormones gibberellic acid (GAs), abscisic acid (ABA) and cytokinins; the carotenoids, chlorophylls and plastoquinones involved in photosynthesis; the ubiquinones required for respiration; and the sterols that influence membrane structure. Monoterpenoids (C 10 ), sesquiterpenoids (C 15 ), diterpenoids (C 20 ) and triterpenoids (C 30 ) are considered to be secondary metabolites (Figure 1). Many of the terpenoids are commercially interesting because of their use as flavors and fragrances in foods and cosmetics (e.g. menthol, nootkatone and sclareol) or because they are important for the quality of agricultural products, such as the flavor of fruits and the fragrance of flowers (e.g. linalool) (Figure 1) [1,2]. In addition, terpenoids can have medicinal properties such as anti-carcinogenic (e.g. Taxol and perilla alcohol), antimalarial (e.g. artemisinin), anti-ulcer, hepaticidal, antimicrobial or diuretic (e.g. glycyrrhizin) activity (Figure 1) [3–7]. The terpenoids have also been shown to be of ecological significance [8,9]. Compounds such as the bitter triterpenoid cucurbitacins and the pungent diterpenoid polygodial have been shown to be involved in insect resistance (Figure 1) [10,11]. Other terpenoid compounds are involved in interactions between plants, between plants and microorganisms, and between plants and arthropod herbivores [e.g. (E,E)-a-farnesene, which is induced in cucumber by spider mite feeding] (Figure 1) [12–14]. The commercial and ecological importance of terpe- noids makes their metabolic engineering an attractive subject for investigation [15]. On the one hand, engineer- ing could lead to the improvement of many input and output traits in crops. These include disease and pest resistance, weed control (e.g. by producing allelopathic compounds), improved fragrance of ornamentals and pollination of seed crops (both by altering floral scent), enhanced aroma of fruits and vegetables, and the production of pharmaceuticals in plants. On the other hand, transgenic plants with modified terpenoid pro- duction could make an important contribution to funda- mental studies of the biosynthesis and regulation of these compounds and their importance in ecological relation- ships. In this article, we describe the latest exciting developments in the engineering of terpenoids in plants. We limit ourselves to the secondary metabolite class of terpenoids as defined above and depicted in Figure 1. Biosynthesis of terpenoids in plants Terpenoids are derived from the mevalonate pathway, which is active in the cytosol, or from the plastidial 2-C- methyl-D-erythritol-4-phosphate (MEP) pathway (Figure 2) [16,17]. Both pathways lead to the formation of the C 5 units isopentenyl diphosphate (IDP) and its allylic isomer dimethylallyl diphosphate (DMADP), the basic terpenoid biosynthesis building blocks. In both compartments, IDP and DMADP are used by prenyl transferases in condensation reactions to produce larger prenyl diphosphates, such as the monoterpene precursor geranyl diphosphate (GDP), the sesquiterpene precursor farnesyl diphosphate (FDP) and the diterpene and C 40 carotenoid precursor geranylgeranyl diphosphate (GGDP) (Figure 2). Condensation of two units of FDP produces squalene, the precursor of triterpenes and sterols. Although there is increasing evidence that there is exchange of intermediates between these compartments [6,18–24], the cytoplasmic mevalonate pathway is gener- ally considered to supply the precursors for the production of sesquiterpenes and triterpenes (including sterols) Corresponding authors: Bouwmeester, H.J. (harro.bouwmeester@wur.nl), Ahar- oni, A. (asaph.aharoni@weizmann.ac.il). Review TRENDS in Plant Science Vol.10 No.12 December 2005 www.sciencedirect.com 1360-1385/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2005.10.005