Cell-Free Synthetic Biology for Pathway Prototyping

Methods in Enzymology
Ashty S Karim, Michael C Jewett

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

Aug 13, 2020·Chembiochem : a European Journal of Chemical Biology·Jonathan W BogartMichael C Jewett
Jun 6, 2019·Methods and Protocols·Hye Jin Lim, Dong-Myung Kim
Jun 17, 2020·Nature Chemical Biology·Ashty S KarimMichael C Jewett
Jun 2, 2020·Frontiers in Bioengineering and Biotechnology·Richard J R KelwickPaul S Freemont
Apr 9, 2020·Frontiers in Bioengineering and Biotechnology·Nadanai LaohakunakornSebastian J Maerkl
Mar 20, 2019·Chemistry : a European Journal·Pasquale Stano
Aug 15, 2020·Frontiers in Bioengineering and Biotechnology·Nadanai Laohakunakorn
Dec 22, 2020·Synthetic Biology·Ashty S KarimMichael C Jewett
Sep 4, 2020·Journal of Industrial Microbiology & Biotechnology·Alexandra A MalicoGavin J Williams
Jun 6, 2020·Metabolic Engineering·William S GrubbeAshty S Karim
Jul 9, 2021·Journal of Agricultural and Food Chemistry·Fu-Xing NiuJian-Zhong Liu
Jul 25, 2019·ACS Synthetic Biology·Amir PandiJean-Loup Faulon
Jan 25, 2020·Journal of Agricultural and Food Chemistry·Fu-Xing NiuJian-Zhong Liu

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