Progress in plant metabolic engineering.

P lants have been estimated to collectively synthesize more than 30,000 different terpenoids, of which many have useful applications in the manufacture of foods, industrial compounds, and pharmaceuticals. Terpenoids are synthesized from the condensation, in a head to tail fashion, of 5-carbon isoprene (or hemiterpene) units. Major terpenoid classes include mono-, sesqui-, and diterpenes, which are mostly secondary metabolites, as well as triand tetraterpenes, which are generally primary metabolites. This large family of compounds includes essential molecules such as carotenoids, gibberellins, abscissic acid and brassinosteroids, sterols, and the phytol chains of chlorophylls, tocopherols, and quinones. However, the vast majority are secondary metabolites, such as the volatile constituents of essential oils, and complex molecules like the anticancer drug paclitaxel that are thought to act as defensive agents (1). The large number of useful terpenoids offers many potentially attractive targets for genetic engineering. In one of the first successful genetic modifications of a plant terpenoid pathway, Mahmoud and Croteau (2) report increasing flux through the monoterpene pathway in mint plants, resulting in an increased essential oil yield. They also improved the quality of the oil by expressing an antisense derivative of the menthofuran synthase gene to down-regulate synthesis of the undesirable constituent menthofuran. Their work builds on a recent major revision in understanding plant terpenoid metabolism and represents a useful example of the state of the art and future directions in metabolic engineering. Until recently, it was thought that the synthesis of terpenoids in higher plants was by a cytosolic route that is derived from mevalonate. However, during the past few years it has become clear that plants also use a parallel plastid pathway that converts pyruvate and glyceraldehyde3phosphate to 1-deoxyxyulose5phosphate (DXP), which is metabolized in a series of steps to isopentenyl diphosphate and dimethylallyl diphosphate—the common precursors of all terpenoids (3). This latter pathway, termed the DXPS pathway, is prevalent in bacteria but has not been found in fungi or most animals. Plants use the mevalonate-dependent pathway to synthesize sesquiterpenes and triterpenes, whereas other major terpenoids derive from the DXPS pathway (3). Because discovery of the plastidial route in plants is relatively recent, little is known of the mechanisms that limit f lux through the DXPS pathway. The gene encoding the first step enzyme 1-deoxy-D-xylulose5-phosphate synthase (DXPS), has been constitutively overexpressed in bacteria and Arabidopsis (4–6). In both cases, increased enzyme activity caused an increase in accumulation of downstream terpenoids, indicating that DXPS is ratelimiting. In Arabidopsis plants, abscisic acid and a-tocopherol were most affected, increasing 4and 2-fold, respectively (6). In mint, it is thought that essential oil monoterpenes, which accumulate in glandular trichomes (Fig. 1), derive from the DXPS pathway (7). Because it is generally undesirable to alter the balance of monoterpenes in the oil, manipulations designed to increase the amount of oil are limited to enzymes downstream of DXPS, but upstream of geranyl diphosphate synthase, the committed step in monoterpene biosynthesis. Mahmoud and Croteau (2) exploited a gene that they had previously isolated, encoding deoxyxylulose phosphate reductoisomerase (DXR), which converts DXP to 2-C-methylerythritol4-phosphate, and constitutes the first committed step in the DXPS pathway of terpenoid biosynthesis (8). They substituted a strong constitutive promoter for the DXR promoter, and introduced the modified DXR gene into peppermint plants. The result was striking: Most transgenic plants accumulated more oil than control plants, with increases of up to 50%. Oil increases were also proportional to DXR activity in the plants and with few exceptions, the chemical composition of the oil was indistinguishable from that of con-