A synthetic biological approach to reconstitution of inositide signaling pathways in bacteria

Abstract Inositide lipid (PIP) and soluble (IP) signaling pathways produce essential cellular codes conserved in eukaryotes. In many cases, deconvoluting metabolic and functional aspects of individual pathways are confoundedby promiscuityand multiplicity of PIP and IP kinases and phosphatases. We report a molecular genetic approach that reconstitutes eukaryoticinositide lipid and soluble pathways in a prokaryotic cellwhich inherently lack inositide kinases and phosphatases in their genome. By expressing synthetic cassettes of eukaryoticgenes, we have reconstructed the heterologousformation of a range of inositide lipids, including PI(3)P, PI(4,5)P2 and PIP3. In addition, we report the reconstruction of lipid-dependent production of inositol hexakisphosphate (IP6). Our synthetic system is scalable, reduces confoundingmetabolic issues, for example it is devoid of inositide phosphatases and orthologous kinases, and enables accurate characterization gene product enzymatic activity and substrate selectivity. This genetically engineered tool is designed to help interpret metabolic pathways and may facilitate in vivo testing of regulators and small molecule inhibitors. In summary, heterologous expressionof inositide pathways in bacteria provide a malleableexperimental platform for aiding signaling biologists and offers new insights into metabolism of these essential pathways.