CHARACTERIZATION OF NOVEL SESQUITERPENOID BIOSYNTHESIS IN TOBACCO EXPRESSING A FUNGAL SESQUITERPENE SYNTHASE

The gene encoding trichodiene synthase (Tri5), a sesquiterpene synthas e from the fungus Fusarium sporotrichioides, was used to transform tob acco (Nicotiana tabacum). Trichodiene was the sole sesquiterpene synth ase product in enzyme reaction mixtures derived from unelicited transf ormant cell-suspension cultures, and both trichodiene and 5-epi-aristo lochene were observed as reaction products following elicitor treatmen t. Immunoblot analysis of protein extracts revealed the presence of tr ichodiene synthase only in transformant cell lines producing trichodie ne. In vivo labeling with [H-3]mevalonate revealed the presence of a n ovel trichodiene metabolite, 15-hydroxytrichodiene, that accumulated i n the transformant cell-suspension cultures. In a trichodiene-producin g transformant, the level of 15-hydroxytrichodiene accumulation increa sed after elicitor treatment. In vivo labeling with [C-14]acetate show ed that the biosynthetic rate of trichodiene and 15-hydroxytrichodiene also increased after elicitor treatment. Incorporation of radioactivi ty from [C-14]acetate into capsidiol was reduced following elicitor tr eatment of a trichodiene-producing transformant as compared with wild type. These results demonstrate that sesquiterpenoid accumulation resu lting from the constitutive expression of a foreign sesquiterpene synt hase is responsive to elicitation and that the farnesyl pyrophosphate present in elicited cells can be utilized by a foreign sesquiterpene s ynthase to produce high levels of novel sesquiterpenoids.