Open AccessMetabolic Dynamics in Escherichia coli-Based Cell-Free Systems
Author(s) -
April M. Miguez,
Yan Zhang,
Fernanda Piorino,
Mark P. Styczynski
Publication year - 2021
Publication title -
acs synthetic biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.156
H-Index - 66
ISSN - 2161-5063
DOI - 10.1021/acssynbio.1c00167
Subject(s) - metabolic pathway , metabolic engineering , metabolism , metabolomics , enzyme , biology , biochemistry , systems biology , protein dynamics , lysis , computational biology , bioinformatics , protein structure
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 preincubation 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.