Biotechnological production of N-heterocycles – blueprints for pharmaceuticals

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Philipp Scheller, Bettina M. Nestl, Bernhard Hauer

University of Stuttgart, Germany

Heterocycles are centerpieces of most natural products and especially in biomolecules like nucleic acids, amino acids, vitamins and alkaloids nitrogen containing rings are overrepresented. This bioactivity is also responsible for N-heterocycles being common backbone elements in the vast majority of small molecule drugs. Hence, they represent ideal precursors for the pharmaceutical industry. However the chemical snytheses of these molecules are hampered by a complex stereochemistry, the dependence on expensive and environmental toxic chemicals as well as low yields. Inspired by the natural biosynthesis of tropane-, piperidine-, pyrrolizidine- and quinolizidine alkaloids, we aim to establish biotechnological production routes resulting in a diverse set of heterocyclic compounds. Although these classes of alkaloids exhibit quite complex and diverse structures, their biosyntheses are initiated with the decarboxylation of only a few amino acids. Using the amino acids arginine, lysine and ornithine different polyamines are generated. They then function as a link between primary and secondary metabolism. The cyclization of these polyamines is a spontaneous reaction, triggered by their oxidation to an aminoaldehyde which will be catalyzed by FAD containing amine oxidases. To gain access to a wide variety of different heterocycles the application and evolution of amine oxidases towards a set of naturally occurring and synthetically modified polyamines is one part of this work. By using methods of directed evolution and rational protein design in combination with high-throughput screening assays based on H2O2 detection, amine oxidase variants and libraries thereof will be created. The assembly of an enzymatic reaction cascade consisting of polyamine modifying enzymes to provide different precursors and the concept of purpose-evolved amine oxidases combined with “tailoring” enzymes for the functionalization of the basic heterocyclic compounds represents a highly flexible approach. Finally the transfer of these enzymes in a living organism will generate artificial pathways for an in vivo production of N-heterocycles.