For the purpose of reprogramming the cellular network in order to achieve high-yield production, we have developed in silico model of the genome-wide metabolism Escherichia coli. Through the application of Metabolic Flux Analysis, we predicted genetic modifications such as deletions and gene expression attenuations that lead to dramatic increases in production levels. Such Systems Biology approaches have resulted in robust production levels leading to potentially commercially viable processes for the synthesis of natural products, in particular ones that derive from malonyl-CoA. We also report the engineering of both positive and negative feedback controls for dynamic tuning of metabolic flux in E. coli. Specifically, we have identified a dual transcriptional regulator that can act either as an activator or a repressor for two different promoters. The level of activation or repression is dependent on the level of intracellular malonyl-CoA. As a proof of concept, we demonstrated that the expression of two reporter proteins can be exclusively switched between the on and off state. By engineering this synthetic malonyl-CoA controller, both a malonyl-CoA source pathway and a malonyl-CoA sink pathway were dynamically modulated so that carbon flux could be efficiently redirected to synthesize our target compounds, fatty acids.
