Abstract
Biological systems provide a sustainable and complimentary approach to synthesizing useful chemical products. Metabolic engineers seeking to establish economically viable biosynthesis platforms strive to increase product titers, rates, and yields. Despite continued advances in genetic tools and metabolic engineering techniques, cellular workflows remain limited in throughput. It may take months to test dozens of unique pathway designs even in a robust model organism, such as Escherichia coli. In contrast, cell-free protein synthesis enables the rapid generation of enzyme libraries that can be combined to reconstitute metabolic pathways in vitro for biochemical synthesis in days rather than weeks. Cell-free reactions thereby enable comparison of hundreds to thousands of unique combinations of enzyme homologs and concentrations, which can quickly identify the most productive pathway variants to test in vivo or further characterize in vitro. This cell-free pathway prototyping strategy provides a complementary approach to accelerate cellular metabolic engineering efforts toward highly productive strains for metabolite production.
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Acknowledgments
This work was supported by the U.S. Department of Energy (DOE) Biological and Environmental Research Division (BER), Genomic Science Program (GSP) for funding of this project under Contract No. DE-SC0018249 and the Office of Energy Efficiency and Renewable Energy Grant DE-EE0008343. B.J.R. is supported by an NDSEG Fellowship (Award ND-CEN-017-095). B.V. was supported by a SNSF Early Postdoc Mobility fellowship (P2SKP3_184036).
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Rasor, B.J., Vögeli, B., Jewett, M.C., Karim, A.S. (2022). Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping. In: Karim, A.S., Jewett, M.C. (eds) Cell-Free Gene Expression. Methods in Molecular Biology, vol 2433. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1998-8_12
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DOI: https://doi.org/10.1007/978-1-0716-1998-8_12
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