Dinglasan and Doktycz, 2023. Synthetic Biology

Cell-free systems can expedite the design and implementation of biomanufacturing processes by bypassing troublesome requirements associated with the use of live cells. In particular, the lack of survival objectives and the open nature of cell-free reactions afford engineering approaches that allow purposeful direction of metabolic flux. The use of lysate-based systems to produce desired small molecules can result in competitive titers and productivities when compared against their cell-based counterparts. However, pathway crosstalk within endogenous lysate metabolism can compromise conversion yields by diverting carbon flow away from desired products. Here, the “block-push-pull” concept of conventional cell-based metabolic engineering was adapted to develop a cell-free approach that efficiently directs carbon flow in lysates from glucose and towards endogenous ethanol synthesis. The approach is readily adaptable, relatively rapid, and allows manipulation of central metabolism in cell extracts. In implementing this approach, a block strategy is first optimized, enabling selective enzyme removal from the lysate to the point of eliminating byproduct forming activity while channeling flux through the target pathway. This is complemented with cell-free metabolic engineering (CFME) methods that manipulate the lysate proteome and reaction environment to push through bottlenecks and pull flux towards ethanol. The approach incorporating these block, push, and pull strategies maximized glucose to ethanol conversion in an E. coli lysate that initially had low ethanologenic potential. A 10-fold improvement in percent yield is demonstrated. To our knowledge, this is the first report of successfully rewiring lysate carbon flux without source strain optimization and to completely transform consumed input substrate to a desired output product in a lysate-based, cell-free system.