A Systems and Synthetic Biology Approach to Engineering Cell-Free Metabolism

Authors: 
Garcia, D. C. - Presenter, Oak Ridge National Laboratory
Dinglasan, J. L., Oak Ridge National Laboratory
Abraham, P. E., Oak Ridge National Laboratory
Hettich, R. L., Oak Ridge National Laboratory
Doktycz, M. J., Oak Ridge National Laboratory
Shrestha, H., Oak Ridge National Laboratory
Cell-free metabolism presents an excellent opportunity to harness the metabolic potential of microbial organisms as the removal from the cellular context enables metabolic engineers to utilize biological systems without the inherent limitations of living organisms. Previous work has demonstrated that crude extracts maintain much of the metabolic potential found inside of the cell and are able to break down feeding substrates such as glucose and energize complex reactions such as transcription and translation using glycolytic intermediates. This work, however, has not fully explored the active metabolism within these systems, as a result, the metabolic potential of the cell-free proteome remains unexplored. Using stable isotope labeling we have analyzed the metabolic flux of a cell-free extract derived from E. coli and used the information in order to engineer E. coli extracts capable of directing flux towards specific metabolites. Using MAGE as a genome engineering platform, deleterious proteins were removed following extract production thus generating a mutant proteome capable of producing conventionally impossible metabolic states with minimal impact on the cell in vivo. This work melds the tools of systems and synthetic biology to create a new paradigm by which precise control can be applied to cell-free metabolic engineering systems.