The tropan-alkaloid scopolamine is an important bulk compound in the semi-synthesis of drugs like Buscopan® or Spiriva®. To date scopolamine is still a plant-based product, extracted from leaves of Duboisia. To secure future demand and to compensate for fluctuations in crop-yield, development of a biotechnological process is of great interest. To prepare for the development of a heterologous production system, several fundamental studies were conducted: Principal pathway analysis. Identification of the genes involved in scopolamine biosynthesis in Duboisia, using degenerated primers based on annotation data of other Solanaceae species. The genes for hyoscyamine 6β-hydroxylase (H6H), littorine mutase/monooxygenase (Cyp80F1), tropine reductase and putrescine N-methyl transferase were found and the key enzymes H6H and Cyp80F1 were quantified using RT-qPCR. cDNA library construction. Using parenchymatic root cells from Duboisia myoporoides, a cDNA library was constructed and sequenced. Contigs were assembled and reconstructed ORFs compared with published sequences from Atropa, Brugmansia and Hyoscyamus for bio-functional characterization, revealing significant differences in the upstream regions of genes like H6H and Cyp80F1. Metabolic and metabolomic profiling. From various organs of Duboisia root-tissue the metabolic profiles of the different tropan-alkaloids were characterised. Quantitative analysis by LC-MS allowed correlation with RT-qPCR data and the main compounds hyoscyamine and scopolamine were found at 5 and 22 μg/mg, respectively. In stem-tissue, scopolamine-glucoside was found, which is likely the form scopolamine is transported in. Metabolomics by Principal Component Analysis of NMR spectra revealed significantly increased levels of inositol, myo-inositol and various amino acids, indicating potentially important roles of these metabolites as pathways-precursors. Metabolic network analysis. Using a combined approach for thermodynamic and stoichiometric analysis of metabolic networks, heterologous production of scopolamine was studied in different organisms. Thermodynamic bottlenecks and major stoichiometric constraints were found, ongoing studies focus on identifying key reactions and engineering targets that influence the viability of a bio-based process.