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Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11799-11803, December 1992 Applied Biological Sciences

Metabolic engineering of medicinal plants: Transgenic Atropa belladonna with an improved alkaloid composition
(scoolamlne/hyoscyamine 61-hydroxylase) DAE-JIN YUN, TAKASHI HASHIMOTO*, AND YASUYUKI YAMADA
Department of Agricultural Chemistry, Faculty of Agriculture, Kyoto University, Kyoto 606-01,Japan

Communicated by Marc Van Montagu, September 15, 1992

The tropane alkaloid scopolamine is a medicABSTRACT inally important anticholinergic drug present in several solanaceous plants. Hyoscyamine 6l-hydroxylase (EC 1.14.11.11) catalyzes the oxidative reactions in the biosynthetic pathway leading from hyoscyamine to scopolamine. We introduced the hydroxylase gene from Hyoscyamus nigerunder the control of the cauliflower mosaic virus 35S promoter into hyoscyaminerich Atropa belladonna by the use of an Agrobacterummediated transformation system. A transgenic plant that constitutively and strongly expressed the transgene was selected, first by screening for kanamycin resistance and then by immunoscreening leaf samples with an antibody specific for the hydroxylase. In the primarytransformant and its selfed progeny that inherited the transgene, the alkloid contents of the leaf and stem were almost exclusively scopolamine. Such metabolically engineered plants should prove useful as breeding materials for obtaining improved medicinal components.

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The use of recombinant DNA technology for the manipulation of metabolic processes in cells promises to provide important contributions to basic science, agriculture, and medicine (1). Secondary metabolism is a particularly attractive target for the improvement of yields of desirable products, without markedly affecting basic cellular functions. The productionof the antibiotic cephalosporin C by a fungal production strain has been improved by giving increased gene dosages of a rate-limiting enzyme (2). Several novel antibiotics have been produced by transferring all or part of their biosynthetic pathways to heterologous host microorganisms (1), as well as by targeted disruption of a biosynthesis gene (3). In plants, flavonoid pigments in ornamentalflowers appear to be the most suitable for genetic modification because the flavonoid biosynthetic pathways and the genes involved are relatively well understood and because any changes in color and pigmentation patterns have potential commercial value (4). A great variety of pharmaceutical and antimicrobial compounds derived from plants also stand to benefit from yield improvement produced bygenetic engineering, but a lack of understanding of the regulation of biosynthetic pathways and the general unavailability of cloned biosynthesis genes severely limit this approach at present. None of the introduced genes that are expected to function in target biosynthetic pathways have produced a considerable increase in the desired phytochemicals in transgenic plants (5-7). The tropane alkaloidshyoscyamine (its racemic form being atropine) and scopolamine are used medicinally as anticholinergic agents that act on the parasympathetic nerve system. Because they differ in their actions on the central nervous system, currently there is a 10-fold higher commercial demand for scopolamine, in the N-butylbromide form, than
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FIG. 1. Biosynthetic pathway from hyoscyamine to scopolamine. Scopolamine is formed from hyoscyamine via 6p-hydroxyhyoscyamine. H6H catalyzes the hydroxylation of hyoscyamine to 6phydroxyhyoscyamine, as well as the epoxidation of...