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P2057
Watchmaker® - Compound Generation by Combinatorial Genetics & Screening in Yeast
Presenter Markus Schwab, Evolva SA, Switzerland
Additional Authors: Stephan van Sint Fiet, Curt Nielsen, Harald Heider, Julia Yakovleva, Sanne Jensen, Philipp Knechtle, Thomas Tange, Anders Hansson, Roberto Archila, Tanja Thybo, Joergen Hansen, Giovanni Salerno, Aurelio Bonavia, Naesby M.
We exploit the power of yeast combinatorial genetics to produce exquisite natural compounds by a strategy based on evolvable yeast artificial chromosomes (eYACS). Thereby a multitude of concatenated enzyme encoding cDNAs derived from diverse plant and animal species in yeast cells are transformed into yeast strains. These enzymes act on endogenous yeast metabolites and, depending on the genetic combination, will then synthesize unknown natural or even novel molecular entities. In a pilot study we introduced into yeast cells cDNAs from diverse organisms encoding for enzymes involved in biosynthesis of carotinoids. These enzymes are fully functional as seen by the yellow color of the colonies derived from genetically engineered yeast cells. Southern Blotting was used to confirm the presence of eYACS that are responsible for b-carotene synthesis in colored colonies.Moreover, we have combined Watchmaker®’s strength of novel compound synthesis with simultaneous screening strategies. Unique screening technologies were implemented to identify the most interesting compounds derived from our genetically engineered yeast. Powerful screens can be achieved by flow cytometry or by the yeast 2-hybrid technology. The principle of a flow cytometry based screening procedure is shown on the poster, as well as a yeast two-hybrid assay for NOD2 oligomerization. NOD2 is a potential drug target in the field of immune response to intracellular bacterial lipopolysaccharides by recognizing the muramyl dipeptide. Mutations in the gene have been associated with Crohn’s disease and Blau’s syndrome. A third assay, set up as a reverse two-hybrid system, is being used for screening compounds that interrupt interaction of mammalian host proteinTSG101 with the Ebola virus VP40 protein. VP40 interacts withTSG101 during viral budding and disruption of this interaction might prevent release of virus particles form host cells.