Abstract
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...Continue Reading
Citations
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