Construction of Enzymes with Synthetic Allosteric Regulation to Control Metabolic Pathways of Escherichia coli

In metabolic engineering strains are created that overproduce a certain product. For that, the production pathway is often released from any transcriptional, translational and post-translational regulation, resulting in a high abundance of enzymes in the production pathway and enzyme variants with feedback-resistance. However, complete dysregulation has several disadvantages: the pathway cannot respond to internal and external perturbations and metabolism of the host is overloaded, resulting in a lowered cellular fitness and reduced growth rates. To circumvent this problem, it is desirable to implement new layers of regulation in the metabolic network and in particular in the overproduction pathway. So far, such regulation has usually been implemented by controlling enzyme abundance. Two-phase processes for instance are dividing a bioprocess in two phases, a growth phase in which a sufficient amount of biomass is accumulated, and a production phase. In this second phase, the expression of enzymes needed for overproduction is induced, often in combination with the introduction of metabolic bottlenecks in competing pathways. However, the regulation of enzyme abundances does not allow fast response at the second or minute time-scale. Especially in large-scale bioreactors fast response is important, because of fluctuating availabilities of nutrients and oxygen caused by insufficient mixing which leads to the formation of microenvironments and dead-zones. Cells in which the overproduction pathway is either dysregulated or regulated only by implemented control of enzyme abundance are not able to adjust their metabolic networks according to fast changing microenvironments, leading to stressed and therefore unproductive strains which might negatively affect the stability and durability of bioprocesses. This highlights the need for faster acting dynamic control of metabolic pathways, for example through enzymes with synthetic allosteric regulation. However, the creation and usage of such enzymes is very challenging. A ...