Terpenoids are widespread in nature and contain hormones, cell constituents and bioactive secondary metabolites. Stable isotope-labeled terpenoids will be powerful tools for biosynthetic studies and functional analysis of unknown biosynthetic genes. We showed enzymatic synthesis and two-dimensional NMR analysis of fully C-13 labeled prenyl diphosphates. Here, we report the construction of multiplex-enzyme cocktails for synthesis of terpenoid hydrocarbons labeled with C-13 isotope. We cloned cDNAs encoding amorphadiene synthase from Artemisia annua L, bifunctional ent-kaurene synthase from Jungermannia subulata, and taxadiene synthase from Taxus cuspidate. These cDNA were expressed in E. coli to produce recombinant proteins. Two enzyme cocktails containing basic cocktail and ent-kaurene synthase or taxadiene synthase were prepared and incubated in the presence of cofactors and substrate as [U-^<13>C_2]acetate. Analysis of the products from each cocktail by GC-MS determined fully-C-13 labeled ent-kaurene and taxadiene, respectively. Another cocktail in which GGDP synthase and diterpene cyclase were removed and amorphadiene synthase was added produced fully-C-13 labeled amorphadiene. Furthermore, only 0.1mg of the labeled amorphadiene was enough to detect C-13 NMR signals. Two-dimensional C-13 NMR analysis (C-C COSY) of labeled amorphadiene at 0.5mg is in progress.
