Genetic engineering has allowed the production of plants with an altered content of secondary metabolites. Because secondary metabolites are important in the defense of plants against pathogens, such engineered plants may show an increase in resistance against pathogens. For example, the expression of a stilbene synthase from Vitis vinifera in tobacco (Nicotiana tabacum) led to the accumulation of stilbenes and thereby to an increased resistance to Botrytis cinerea, providing direct evidence of the role of stilbenes as phytoalexins (Hain et al., 1993). In other experiments the accumulation of secondary metabolites, which are already produced in the untransformed plants, has been increased by the overexpression of structural genes encoding biosynthetic enzymes. Expression of hyoscyamine 6-hydroxylase in Atropa belladonna plants, for example, led to a strong increase of scopolamine production in the transgenic plants (Yun et al., 1992), and expression of a bacterial Lys decarboxylase in tobacco increased the production of the diamine cadaverine (Fecker et al., 1993). Such experiments may prove useful for the production of The commercial production of a pharmaceutical substance by plant cell culture has already been realized on an industrial scale in the case of shikonin, a naphthoquinone pigment with antibacterial, antiphlogistic, and woundhealing properties that is obtained from cell cultures of Lithospermum erythrorhizon (Tabata and Fujita, 1985). Shikonin is biosynthetically derived from 4HB and geranylpyrophosphate (Heide and Tabata, 1987). Using feeding experiments with [1,7-13C,Jshikimic acid ( Fig. 1; Heide et al., 1989), we have shown that the production of 4HB in these cell cultures proceeds exclusively via phenylpropanoid intermediates, and it has been proposed that most benzoic acids as well as ubiquinones are derived from the phenylpropanoid pathway in plants (Pennock and Threlfall, 1983). Furthermore, the conversion of the phenylpropanoid precursors to 4HB was recently characterized (Loscher and Heide, 1994)' showing that the reaction sequence from chorismate to 4HB in plants involves up to 10 successive enzymatic reaction steps in cell cultures of Lithospermum erythrorhizon.Escherichia coli, on the other hand, possesses a simpler biosynthetic route to 4HB, which involves the direct conversion of chorismate to 4HB by CPL (Fig. lb). The cloning of ubiC, the gene encoding CPL, was recently reported by our group (Siebert et al., 1992(Siebert et al., , 1994 and by Nichols and Green (1992). The protein was overexpressed, purified, characterized, and shown to be a soluble protein of 19 kD.It has a K, value for chorismate of 6.1 PM, a pH optimum at 7.5, and does not require cofactors.In this study we have expressed the ubiC gene in tobacco, thereby introducing a single-step process for the production of 4HB in plants. Chorismate, the substrate of the ubiC gene product, is an intermediate of the shikimate pathway. In plants this pathway is localized in the plastid, and the existence of an additional shikimat...

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The carbon skeleton of the naphthoquinone pigment shikonin, which is produced in Lithospermum erythrorhizon Sieb. et Zucc. cell-suspension cultures, is partly derived from the isoprenoid biosynthetic pathway. The requirement of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR, EC 1.1.1.34), a key enzyme of the mevalonate route to isoprenoids, for shikonin synthesis was investigated. Conserved regions of sequences from plant HMGR genes were used to design polymerase chain reaction (PCR) primers for the cloning of a cDNA fragment from L. erythrorhizon. The resulting 443-bp clone was used as a probe for Northern analyses and hybridized to an mRNA of approx. 2.5 kb. Under shikonin-producing conditions, microsomal HMGR enzyme activity as well as mRNA level closely correlated with the accumulation of shikonin derivatives. White light, which inhibits shikonin formation, was shown to strongly suppress HMGR gene expression. The results presented here indicate that HMGR plays a significant role in the regulation of shikonin biosynthesis and that the control appears to act at the transcriptional level.

Chorismate pyruvatc-lyasc activity was detcctcd in uxtracls ol'Esc/wticf~in coli. 4.Hydroxybcnzoate was identified as the product of the cnzymntic reaction by chemical dcrivakation and X-MS analysis. The u&C gene, coding for the chorismate pyruvate-lyass, was cloned and sequenced. The molecular weight of the gene producl was calculated as 18.776 Da and confimxd by expression of the protein in &. cd' minicells. The ubiA gene, coding for Abe 4-hydroxybcnzoote octaprenyl transferase, was identified by sequence homology and complcmcntsGon of a ubiA_ stwin. It is located directly downstream of ubiC in a typical apron structure.

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