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Abstract
Cell-free biology provides a unique opportunity to study complicated cellular systems such as protein synthesis and metabolism. Since no cell wall is present, the reaction environment can be directly controlled and easily sampled. In addition, the cell extract provides a stable catalyst system that is not modified by cellular responses. Cell-free protein synthesis (CFPS) has the potential for higher productivity than in vivo systems because the cellular resources are directed toward the production of a single protein. However, widespread use of CFPS has not been adopted in part because of high reagent costs and short reaction times caused by substrate instabilities.
In this work, we investigate the metabolism associated with major classes of compounds during CFPS, including amino acids, organic acids, and nucleotides. By understanding this metabolism, we address the challenges of substrate instability and high reagent cost. Amino acid supply is stabilized through genetic modification of the source strain used to make cell extract. By deleting five genes, we remove unwanted enzymatic activities and produce an active extract that maintains stable amino acid concentrations. Energy supply limitations are also addressed. Since glucose is the preferred carbon and energy source of the biotechnology industry, we sought conditions for which glycolysis was activated to supply the energy needs of the protein synthesis reaction. Stabilizing pH and removing phosphate limitations allow glucose utilization. Glucose is two orders of magnitude less expensive than traditional energy sources, such as phosphoenolpyruvate. We further reduce reagent costs by substituting nucleoside monophosphates (NMPs) for nucleoside triphosphates (NTPs). NMPs are quickly converted to NTPs with no reduction in protein synthesis yields or energy charge. Finally, metabolite measurements are used to define a model of cell-free metabolism solved with dynamic flux balance analysis. The model allows a broad view of cell-free metabolism and gives insights into reaction flux and metabolite data that we cannot measure experimentally.
Overall, this investigation has contributed to cell-free biology by increasing substrate stability, decreasing reagent costs, and developing a better understanding of cell-free metabolism. These results are important for improving CFPS to enable a variety of applications.
