Cell-Free Synthetic Biology for Pathway Prototyping

Engineering biological systems for the production of biofuels and bioproducts holds great potential to transform the bioeconomy, but often requires laborious, time-consuming design-build-test cycles. For decades cell-free systems have offered quick and facile approaches to study enzymes with hopes of informing cellular processes, mainly in the form of purified single-enzyme activity assays. Over the past 20 years, cell-free systems have grown to include multienzymatic systems, both purified and crude. By decoupling cellular growth objectives from enzyme pathway engineering objectives, cell-free systems provide a controllable environment to direct substrates toward a single, desired product. Cell-free approaches are being developed for prototyping and for biomanufacturing. In prototyping applications, the idea is to use cell-free systems to test and optimize biosynthetic pathways before implementation in live cells and scale-up. We present a detailed method for the generation of crude lysates for cell-free pathway prototyping, mix-and-match cell-free metabolic engineering using preenriched lysates, and cell-free protein synthesis driven cell-free metabolic engineering. The cell-free synthetic biology methods described herein are generalizable to any biosynthetic pathway of interest and provide a powerful approach to building pathways in crude lysates for the purpose of prototyping. The foundational principle of the presented approach is that we can construct discrete metabolic pathways through modular assembly of cell-free lysates containing enzyme components produced by overexpression in the lysate chassis strain or by cell-free protein synthesis (in vitro production). Overall, the modular and cell-free nature of our pathway prototyping framework is poised to facilitate multiplexed, automated study of biosynthetic pathways to inform systems-level cellular design.