Enzymatic reaction networks are unique in that one can operate a large number of reactions under the same set of conditions concomitantly in one pot, but the nonlinear kinetics of the enzymes and the resulting system complexity have so far defeated rational design processes for the construction of such complex cascade reactions. Here we demonstrate the forward design of an in vitro 10-membered system using enzymes from highly regulated biological processes such as glycolysis. For this, we adapt the characterization of the biochemical system to the needs of classical engineering systems theory: we combine online mass spectrometry and continuous system operation to apply standard system theory input functions and to use the detailed dynamic system responses to parameterize a model of sufficient quality for forward design. This allows the facile optimization of a 10-enzyme cascade reaction for fine chemical production purposes.
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