A lysate proteome engineering strategy for enhancing cell-free metabolite production.

Cell-free systems present a significant opportunity to harness the metabolic potential of diverse organisms. Removing the cellular context provides the ability to produce biological products without the need to maintain cell viability and enables metabolic engineers to explore novel chemical transformation systems. Crude extracts maintain much of a cell's capabilities. However, only limited tools are available for engineering the contents of the extracts used for cell-free systems. Thus, our ability to take full advantage of the potential of crude extracts for cell-free metabolic engineering is constrained. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective depletion from crude extracts so as to specifically direct chemical production. Specific edits to central metabolism are possible without significantly impacting cell growth. Selective removal of pyruvate degrading enzymes resulted in engineered crude lysates that are capable of up to 40-fold increases in pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to showcase the effectiveness of cell-free metabolic engineering for applications like bioprototyping and bioproduction.