In vitro prototyping and rapid optimization of biosynthetic enzymes for cell design

Ashty S. Karim, Quentin M. Dudley, Alex Juminaga, Yongbo Yuan, Samantha A. Crowe, Jacob T. Heggestad, Shivani Garg, Tanus Abdalla, William S. Grubbe, Blake J. Rasor, David N. Coar, Maria Torculas, Michael Krein, Fung Min (Eric) Liew, Amy Quattlebaum, Rasmus O. Jensen, Jeffrey A. Stuart, Sean D. Simpson, Michael Köpke*, Michael C. Jewett*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

136 Scopus citations


The design and optimization of biosynthetic pathways for industrially relevant, non-model organisms is challenging due to transformation idiosyncrasies, reduced numbers of validated genetic parts and a lack of high-throughput workflows. Here we describe a platform for in vitro prototyping and rapid optimization of biosynthetic enzymes (iPROBE) to accelerate this process. In iPROBE, cell lysates are enriched with biosynthetic enzymes by cell-free protein synthesis and then metabolic pathways are assembled in a mix-and-match fashion to assess pathway performance. We demonstrate iPROBE by screening 54 different cell-free pathways for 3-hydroxybutyrate production and optimizing a six-step butanol pathway across 205 permutations using data-driven design. Observing a strong correlation (r = 0.79) between cell-free and cellular performance, we then scaled up our highest-performing pathway, which improved in vivo 3-HB production in Clostridium by 20-fold to 14.63 ± 0.48 g l−1. We expect iPROBE to accelerate design–build–test cycles for industrial biotechnology. [Figure not available: see fulltext.]

Original languageEnglish (US)
Pages (from-to)912-919
Number of pages8
JournalNature Chemical Biology
Issue number8
StatePublished - Aug 1 2020

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology


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