Principal Studies on the Scopolamine Biosynthesis in Duboisia Spp. for Heterologous Reconstruction of Tropane Alkaloid Biosynthesis
The alkaloid scopolamine is an important bulk compound in the semi-synthesis of drugs for clinical medicines like Buscopan or Spiriva. To date scopolamine is still a plant-based product; the majority is obtained via extraction from Duboisia species. As demand is expected to increase in the future and to compensate for fluctuations in crop yield of the medicinal plants, development of a biotechnological process is becoming of great interest. In order to assemble a heterologous pathway fundamental understanding of the merging (acetate- and shikimate-) pathways, including biosynthetic genes, their transcription and regulation, is crucial. Therefore several fundamental studies were conducted: i) Analysis of the principal pathway and most important enzymes for their expression and biochemical profile, ii) Construction of a cDNA library from cytosolic root cells of Duboisia species, iii) Analysis of metabolic and metabolomic profiles of Duboisia species, iv) In silico investigation of a heterologous production system utilising metabolic network modelling.
Principal pathway analysis. Based on database annotation data of Solanaceae species primers were designed for all relevant genes involved in the scopolamine biosynthesis, starting with the first precursors in the acetate- and shikimate-pathway through to tropanol and phenyllactic acid, respectively. Despite the unavailability of comprehensive information on the Duboisia genome, we were able to identify and detect the genes for H6H, Cyp80F1, tropine reductase and putrescine N-methyltransferase using degenerated primer pairs. In addition, the key enzymes H6H and Cyp80F1 were quantified using RT-PCR.
cDNA library construction. Using parenchymatic root cells from Duboisia myoporoides, a cDNA library was constructed and sequenced (Ilumina 454). Contigs were assembled and full gene ORFs reconstructed and compared with database published sequences from other Solanacea plants in the genus of Atropa, Hyoscyamus and Burgmansia. Obtained genes, like H6H and Cyp80F1, were compared and aligned for bio-functional characterisation. Based on sequencing data significant base differences were determined in the upstream region of the genes.
Metabolic and metabolomic profiling. From various organs of root-tissue the metabolic profile of eight tropan alkaloids including putrescine, tropanol, hyoscyamine, 6-OH-hyoscyamine, and scopolamine was characterised. Quantitative analysis was performed by means of LC-MS to allow a correlation with RT-PCR results. The main compounds hyoscyamine and scopolamine were found to be in the range of 5 to 22 μg/mg, respectively. Interestingly, scopolamine glucoside was found in stem tissue, which is likely the form of transported scopolamine. In-detail metabolome analysis was conducted based on NMR studies: recorded NMR resonances were subject to Principal Component Analysis. A statistical analysis revealed that various amino acids as well as inositol and myo-inositol were significantly increased indicating potentially important roles of these as precursors to supply flux to the product pathways.
Metabolic network analysis. Using a novel tool for network-embedded thermodynamic analysis of elementary flux modes, a heterologous tropane-alkaloid pathway was studied in the cell envelope. This identified thermodynamic bottlenecks and major stoichiometric constraints. Ongoing studies focus on identifying key reactions and engineering targets that influence the viability of a bio-based process.