Cell-Free Protein Synthesis for High-Throughput Biosynthetic Pathway Prototyping

Blake J. Rasor, Bastian Vögeli, Michael C. Jewett*, Ashty S. Karim

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

6 Scopus citations


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.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Number of pages17
StatePublished - 2022

Publication series

NameMethods in Molecular Biology
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029


  • Biosynthetic pathways
  • Cell-free
  • Enzyme assay
  • High-throughput screening
  • In vitro
  • Metabolic engineering
  • TX-TL

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics


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