A cell-free framework for rapid biosynthetic pathway prototyping and enzyme discovery

Ashty S. Karim, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

157 Scopus citations


Speeding up design-build-test (DBT) cycles is a fundamental challenge facing biochemical engineering. To address this challenge, we report a new cell-free protein synthesis driven metabolic engineering (CFPS-ME) framework for rapid biosynthetic pathway prototyping. In our framework, cell-free cocktails for synthesizing target small molecules are assembled in a mix-and-match fashion from crude cell lysates either containing selectively enriched pathway enzymes from heterologous overexpression or directly producing pathway enzymes in lysates by CFPS. As a model, we apply our approach to n-butanol biosynthesis showing that Escherichia coli lysates support a highly active 17-step CoA-dependent n-butanol pathway in vitro. The elevated degree of flexibility in the cell-free environment allows us to manipulate physiochemical conditions, access enzymatic nodes, discover new enzymes, and prototype enzyme sets with linear DNA templates to study pathway performance. We anticipate that CFPS-ME will facilitate efforts to define, manipulate, and understand metabolic pathways for accelerated DBT cycles without the need to reengineer organisms.

Original languageEnglish (US)
Pages (from-to)116-126
Number of pages11
JournalMetabolic Engineering
StatePublished - Jul 1 2016


  • Biosynthetic pathways
  • Cell-free metabolic engineering (CFME)
  • Cell-free protein synthesis (CFPS)
  • Design-build-test (DBT)
  • N-butanol
  • Synthetic biology

ASJC Scopus subject areas

  • Applied Microbiology and Biotechnology
  • Bioengineering
  • Biotechnology


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