Abstract
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.
Highlights
Driven by the prospect of biological systems that can be manipulated, the application of synthetic biology tools to in vitro environments offers a promising approach to harnessing an organism’s rich metabolic potential (Guterl et al, 2012)
We describe the use of genome engineering, Multiplex Automated Genomic Engineering (MAGE), to enable the removal of particular proteins from crude extracts for cell-free metabolic engineering (CFME). 6xHis-tags are incorporated into proteins that are expected to consume pyruvate and are used for the affinity-based depletion of these proteins following cell lysis
Pyruvate dehydrogenase (Pdh), responsible for pyruvate flux in aerobic conditions, is expected to be expressed under these conditions as respiratory metabolism is reportedly active in S30 lysates (Foshaget al., 2018)
Summary
Driven by the prospect of biological systems that can be manipulated, the application of synthetic biology tools to in vitro environments offers a promising approach to harnessing an organism’s rich metabolic potential (Guterl et al, 2012). Cell-free systems use cytoplasmic components, devoid of genetic material and membranes, as a means of producing complex chemical transformations. While living cells require membranes, growth substrates, and biochemical regulation, in vitro systems sidestep these barriers to manipulation and present an opportunity to explicitly define a system for creating novel proteins and metabolites (Shin and Noireaux, 2012). In this way, cell-free metabolic engineering (CFME) can use the organism’s existing biochemical functions and further combine these capabilities with heterologous pathways to produce chemical precursors, biofuels, and pharmaceuticals. Constructing complex, multistep pathways require significant development and upfront costs as utilizing purified proteins at scale remains costly (Korman et al, 2017)
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