Diterpene synthesis in Stevia rebaudiana: recruitment and up-regulation ofkey enzymes from the gibberellin biosynthetic pathway

Stevia rebaudiana Bertoni leaves accumulate a mixture of at least eight dif ferent glycosides derived from the tetracyclic diterpene steviol. These nat ural products taste intensely sweet and have similar biosynthetic origins t o those of gibberellic acid (GA). The initial steps leading to the formatio n of GA result from the two-step cyclization of geranylgeranyl diphosphate (GGDP) to (-)-kaurene via the action of two terpene cyclases (-)-copalyl di phosphate synthase (CPS) and (-)-kaurene synthase (KS). Steviol biosynthesi s probably uses the same mechanism although the genes and enzymes from S, r ebaudiana that are involved in the cyclization of GGDP have not been charac terized. We have isolated both the CPS and KS genes from S, rebaudiana and found that recombinant CPS and KS were catalytically active, suggesting tha t the CPS and KS genes participate in steviol biosynthesis. The genes codin g for CPS and KS are usually present in single copies in most plant species and their expression is normally low and limited to rapidly growing tissue s. The KS gene has been duplicated in the S. rebaudiana genome and both the KS and CPS genes are highly expressed in mature leaves, a pattern opposite to that found with GA biosynthesis. This pattern may, at least in part, le ad to temporal and spatial separation of GA and steviol biosynthesis and pr obably helps to prevent over-expression from interfering with normal GA met abolism. Our results show that CPS and KS are part of the steviol glycoside biosynthetic pathway and that Stevia rebaudiana has recruited two genes to secondary metabolism from a highly regulated pathway involved in hormone b iosynthesis.