Expression of a Fungal Sesquiterpene Cyclase Gene in Transgenic Tobacco

Abstract

The complete coding sequence for the trichodiene synthase gene from Fusarium sporotrichioides was introduced into tobacco (Nicotiana tabacum) under the regulation of the cauliflower mosiac virus 35S promoter. Expression of trichodiene synthase was demonstrated in the leaves of transformed plants. Leaf homogenates incubated with [3H]famesyl pyrophosphate produced trichodiene as a major product. Trichodiene was detected in the leaves of a transformed plant at a level of 5 to 10 nanograms per gram fresh weight. The introduction of a fungal sesquiterpene cyclase gene into tobacco has resulted in the expression of an active enzyme and the accumulation of low levels of its sesquiterpenoid product.Sesquiterpene cyclases participate in the biosynthesis of most sesquiterpenoids. This group of enzymes converts the isoprenoid pathway intermediate, FPP,2 into an estimated 100 to 200 different cyclic sesquiterpenoids (2). Information concerning the properties of sesquiterpene cyclases has been reported for isolated or partially purified enzyme preparations from plants (4, 13), fungi (9, 11), and a streptomycete (1). Comparisons among these enzymes indicate that they are mechanistically similar but differ with respect to properties such as molecular weight and subunit composition.Plants, fungi, insects, and marine invertebrates all produce large numbers of structurally distinct sesquiterpenoids. In many cases, distantly related organisms accumulate the same sesquiterpene cyclase product or produce sesquiterpenoids derived from the same parent compound. This means that genes coding for any particular sesquiterpene cyclase might be available from several sources.The modification of plant sesquiterpenoid biosynthesis could result in altered resistance to disease and insect pests. Potential targets of such efforts include the production of sesquiterpenoid phytoalexins within the Solanaceae (16) (12). Trichodiene synthase is the enzyme involved in trichodiene biosynthesis. A gene encoding trichodiene synthase has been isolated from Fusarium sporotrichioides (8) and expressed in Escherichia coli. Expression of trichodiene synthase in E. coli resulted in the production of active enzyme and in the accumulation of trichodiene (10).The expression of foreign sesquiterpene cyclase genes in plants represents one possible approach for altering sesquiterpenoid biosynthesis. To determine the feasibility of this approach, we introduced the trichodiene synthase gene into tobacco. Expression in tobacco may provide insights into both the regulation of sesquiterpenoid biosynthesis and the role of these compounds in disease resistance. MATERIALS AND METHODS Transformation of TobaccoThe BamHI fragment from pTS37-4A(470-529), containing the complete coding sequence for the trichodiene synthase gene of Fusarium sporotrichioides (10), was inserted into the plasmid, pBI221 (Clontech3), from which the ,B-glucuronidase-coding sequence had been deleted (SmaI-SstI fragment EXPRESSION OF TRICHODIENE SYNTHASE IN TOBACCOmosaic virus 35S promoter, a u...

Terpenoid Metabolism in Wild-Type and Transgenic Arabidopsis Plants[W]

“…In contrast to transgenic plants overexpressing monoterpene synthases, the low production of transgenic plants overexpressing sesquiterpene synthases seems to be a general phenomenon. Just as we have shown for germacrene A synthase expression in Arabidopsis (Figure 10), other authors have reported low production of sesquiterpenes from transgenic tobacco plants overexpressing trichodiene synthase and amorpha-4,11-diene synthase (Hohn and Ohlrogge, 1991;Wallaart et al, 2001). The pool of FDP in the cytosol also is used for the production of triterpenes and sterols in the same compartment.…”

“…Two sesquiterpene synthase genes also have been expressed in transgenic plants. Tobacco plants were transformed by Hohn and Ohlrogge (1991) with a fungal trichodiene synthase and by Wallaart et al (2001) with the Artemisia annua amorpha-4,11-diene synthase. In both cases, only very low levels of the expected sesquiterpenoids were detected.…”

Metabolic Engineering of Terpenoid Biosynthesis in Plants

“…In conclusion, in many studies it was demonstrated that it is feasible to engineer several steps of the monoterpenoid pathway. However, attempts to engineer sesquiterpenes in plants using terpene synthases resulted in only low level production (Hohn and Ohlrogge, 1991;Wallaart et al, 2001).…”

Metabolic engineering of medicinal plants: transgenic Atropa belladonna with an improved alkaloid composition.

“…A great variety of pharmaceutical and antimicrobial compounds derived from plants also stand to benefit from yield improvement produced by genetic 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)(6)(7). The tropane alkaloids hyoscyamine (its racemic form being atropine) and scopolamine are used medicinally as anticholinergic agents that act on the parasympathetic nerve system.…”