Engineering robust control of two-component system phosphotransfer using modular scaffolds

Synthetic biology applies engineering principles to facilitate the predictable design of biological systems. Biological systems composed of modular parts with clearly defined interactions are generally easier to manipulate than complex systems exhibiting a large number of subtle interactions. However, recreating the function of a naturally complex system with simple modular parts can increase fragility. Here, inspired by scaffold-directed signaling in higher organisms, we modularize prokaryotic signal transduction to allow programmable redirection of phosphate flux from a histidine kinase to response regulators based on targeting by eukaryotic protein–protein interaction domains. Although scaffold-directed colocalization alone was sufficient to direct signaling between components, this minimal system suffered from high sensitivity to changing expression levels of each component. To address this fragility, we demonstrate how to engineer autoinhibition into the kinase so that phosphotransfer is possible only upon binding to the scaffold. This system, in which scaffold performs the dual functions of activating this autoinhibited kinase and directing flux to the cotargeted response regulator, was significantly more robust to varying component concentrations. Thus, we demonstrate that design principles inspired by the complex signal-transduction pathways of eukaryotes may be generalized, abstracted, and applied to prokaryotes using well-characterized parts.