PT - JOURNAL ARTICLE AU - April M. Miguez AU - Yan Zhang AU - Fernanda Piorino AU - Mark P. Styczynski TI - Metabolic Dynamics in <em>Escherichia coli</em>-based Cell-Free Systems AID - 10.1101/2021.05.16.444339 DP - 2021 Jan 01 TA - bioRxiv PG - 2021.05.16.444339 4099 - http://biorxiv.org/content/early/2021/05/17/2021.05.16.444339.short 4100 - http://biorxiv.org/content/early/2021/05/17/2021.05.16.444339.full AB - The field of metabolic engineering has yielded remarkable accomplishments in using cells to produce valuable molecules, and cell-free expression (CFE) systems have the potential to push the field even further. However, CFE systems still face some outstanding challenges, including endogenous metabolic activity that is poorly understood yet has a significant impact on CFE productivity. Here, we use metabolomics to characterize the temporal metabolic changes in CFE systems and their constituent components, including significant metabolic activity in central carbon and amino acid metabolism. We find that while changing the reaction starting state via lysate pre-incubation impacts protein production, it has a comparatively small impact on metabolic state. We also demonstrate that changes to lysate preparation have a larger effect on protein yield and temporal metabolic profiles, though general metabolic trends are conserved. Finally, while we improve protein production through targeted supplementation of metabolic enzymes, we show that the endogenous metabolic activity is fairly resilient to these enzymatic perturbations. Overall, this work highlights the robust nature of CFE reaction metabolism as well as the importance of understanding the complex interdependence of metabolites and proteins in CFE systems to guide optimization efforts.Abstract FigureCompeting Interest StatementThe authors have declared no competing interest.