In vitro Degradation
Upscale metabolic engineering has highest potential achieving the production of biofuels, therapeutically products and speciality chemicals with genetically modified organisms. Still, time-consuming purification runs after fermentation and the limitations of in vivo reactions like toxicity of accumulated intermediates due to slow turnover rates of pathway enzymes or the influence of key enzymes on host metabolism can disfavor bioreactors. In our approach we tend to solve these problems in vitro by immobilizing all needed pathway enzymes in close proximity on the bioreactor surface using a protein scaffold. In consequence, pathway enzymes remain in our in vitro bioreactor during purification and can be reused in the next fermentation. As a proof of principle we use the degradation of xylan, a group of hemicellulose, to β-D-xylose, which can be used as substrate for biosynthesis of all three targeted monomers for the synthesis of a photopolymer. The model degradation pathway is immobilized on a silica surface by using a fusion protein consisting of the protein scaffold by iGEM TU Darmstadt 2014 and the Si4-tag by iGEM Leeds 2013. In addition we tested the functionality of Polyester Degradation for variation of used waste inside of the bioreactor.