dCas9 as a tool for metabolic pathway assembly

The synthesis of novel biochemicals will in some cases require a changed substrate flow in the pathways and in most cases optimization to increase yield will be required. Spatial organization can help substrates flow between proteins. Recently, it has been suggested to use RNA as scaffolds to spatially arrange multi-enzymatic pathways. A more tuneable DNA anchoring is, in principle, offered by the CRISPR/cas system. Cas9 nuclease from the type II CRISPR-Cas system has been turned into a programmable DNA-binding protein by introducing mutations in some domains. This Cas9 nuclease mutant named 'dead' Cas9 (dCas9) allows for targeted DNA binding without DNA editing. This method allows tuning in genetic expression. Fusion proteins of dCas9 protein and target proteins of a specific pathway will be targeted using sgRNAs towards a defined DNA scaffold that allows the spatial arrangement of the pathway. As a proof of concept we want to produce fluorescence by bringing together the two parts of the Yellow Fluorescent Protein (YFP). After that we will be able to move to a more attractive and complicated pathway. We expect to obtain an increased yield of the enzymatic pathway that can be industrially applicable. We will also test the method in a standardized manner across different strain species.