The former point is addressed by pre-engineering cellular enzymatic networks such that the desired reaction networks in the resulting cell free extracts become more and more insulated. By removing small molecules from the system, this insulation can be advanced already rather far, but problems with enzymes essential for growth remain, in addition to other enzymes that interfere with the insulation of either the metabolic network or additional functions such as cofactor regeneration. These problems can be addressed by developing strategies to selectively eliminate enzymes from a cell free extract after production, such as selective proteolytic degradation.
In order to address the latter point, it is imperative to implement the right analytical tools to study the time-behavior of the molecular network in sufficient detail. We have worked towards a real-time MS-method that allows tracking the effect of perturbations to the network in high data-density and frequency. This in turn allows on-line optimization of enzyme networks, such as the upper part of glycolysis.