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 limited. Here, we employ Multiplex Automated Genomic Engineering (MAGE) to tag proteins for selective removal 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 are demonstrated that result in engineered crude lysates that are capable of 10 to 20-fold increases of pyruvate production when compared to the non-engineered extract. The described approach melds the tools of systems and synthetic biology to develop cell-free metabolic engineering into a practical platform for both bioprototyping and bioproduction. Highlights A novel method of engineering crude cell lysates for enhancing specific metabolic processes is described. Multiplex Automated Genomic Engineering (MAGE) can be used to engineer donor strains for improving cell-free metabolite production with minimal impact on cell-growth. The described lysate engineering strategy can specifically direct metabolic flux and create metabolic states not possible in living cells. Pooling of the central precursor pyruvate was significantly improved through use of this lysate proteome engineering strategy.